Methods for modulating an immune response

ABSTRACT

The present disclosure provides methods of modulating an immune response in an individual. The present disclosure provides methods of treatment. The present disclosure provides methods comprising administering a multimeric polypeptide (synTac) and an immune checkpoint inhibitor to an individual. The present disclosure provides methods comprising administering a multimeric polypeptide (synTac) to an individual who is undergoing treatment with immune checkpoint inhibitor.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/471,832, filed Mar. 15, 2017, and of U.S. ProvisionalPatent Application No. 62/521,009, filed Jun. 16, 2017, whichapplications are incorporated herein by reference in their entirety.

INTRODUCTION

An adaptive immune response involves the engagement of the T cellreceptor (TCR), present on the surface of a T cell, with a small peptideantigen non-covalently presented on the surface of an antigen presentingcell (APC) by a major histocompatibility complex (MHC; also referred toin humans as a human leukocyte antigen (HLA) complex). This engagementrepresents the immune system's targeting mechanism and is a requisitemolecular interaction for T cell modulation (activation or inhibition)and effector function. Following epitope-specific cell targeting, thetargeted T cells are activated through engagement of costimulatoryproteins found on the APC with counterpart costimulatory proteins the Tcells. Both signals—epitope/TCR binding and engagement of APCcostimulatory proteins with T cell costimulatory proteins—are requiredto drive T cell specificity and activation or inhibition. The TCR isspecific for a given epitope; however, the costimulatory protein notepitope specific and instead is generally expressed on all T cells or onlarge T cell subsets.

SUMMARY

The present disclosure provides methods of modulating an immune responsein an individual. The present disclosure provides methods of treatment.The present disclosure provides methods comprising administering amultimeric polypeptide (synTac) and an immune checkpoint inhibitor to anindividual. The present disclosure provides methods comprisingadministering a multimeric polypeptide (synTac) to an individual who isundergoing treatment with immune checkpoint inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D schematically depict various embodiments of a T-cellmodulatory multimeric polypeptide. In these embodiments, disulfide bondsare formed between MHC (e.g., HLA) polypeptides present in separatepolypeptides.

FIG. 2A-2Q provide an amino acid sequence of wild-type human IL-2 (FIG.2A); and amino acid sequences of variant IL-2 polypeptides (FIG. 2B-2Q).

FIG. 3A-3C provide amino acid sequences of IL-2 receptor alpha chain(FIG. 3A), beta chain (FIG. 3B), and gamma chain (FIG. 3C).

FIG. 4A-4C provide amino acid sequences of immunoglobulin Fcpolypeptides.

FIG. 5A-5C provide amino acid sequences of human leukocyte antigen (HLA)Class I heavy chain polypeptides. Signal sequences are underlined.

FIG. 6 provides a multiple amino acid sequence alignment of beta-2microglobulin (β2M) precursors (i.e., including the leader sequence)from Homo sapiens (NP_004039.1; SEQ ID NO:95), Pan troglodytes(NP_001009066.1; SEQ ID NO:195), Macaca mulatta (NP_001040602.1; SEQ IDNO:96), Bos taurus (NP_776318.1; SEQ ID NO:97) and Mus musculus(NP_033865.2; SEQ ID NO:98). Amino acids 1-20 are a signal peptide.

FIG. 7A-7B depict production of IL-2/synTacs (“Cue-IL-2-a” andCue-IL-2-b”) of the present disclosure following transient transfection.FIG. 7A depicts unpurified yields; FIG. 7B depicts purified product.

FIG. 8A-8B depict production of IL-2/synTacs of the present disclosure,in which the IL-2 polypeptide is present on the light chain (thepolypeptide chain with the light chain (e.g., β2M) of an MHC Class Imolecule) or on the heavy chain (the polypeptide chain with the heavychain of an MHC Class I molecule).

FIG. 9 depicts the expression level of IL-2/syn-Tacs, in which the IL-2is wild-type (wt), or comprises various combinations of F42A, D20K,Q126A, E15A, Y45A, and H16A.

FIG. 10 depicts expression of IL-2/synTacs of the present disclosure, inwhich the IL-2 is present in one copy (1×), two copies (2×) or threecopies (3×) in the synTac.

FIG. 11 depicts in vitro stimulation of antigen-specific CD8⁺ T cellsand non-specific CD8⁺ T cells by an IL-2/synTac of the presentdisclosure, where the IL-2 variant comprising F42A and H16Asubstitutions is present in the synTac in two copies.

FIG. 12 depicts IL-2/synTac binding to specific (lymphocyticchoriomeningitis virus; LCMV) or non-specific (OTI; recognizingovalbumin) CD8⁺ T cells.

FIG. 13 depicts IL-2/synTac-mediated signaling in antigen-specific(LCMV) or non-specific (BL6) CD8⁺ T cells.

FIG. 14A-14F depict the percent phospho-signal transducer and activatorof transcription 5 (pSTAT5)-positive cells following stimulation of CD8⁺antigen-specific (LCMV) or non-specific (BL6) cells with IL-2/synTacs ofthe present disclosure at various IL-2/synTac concentrations.

FIG. 15 depicts in vivo activity of an IL-2/synTac of the presentdisclosure. The left panel depicts the fold change in the number ofantigen-specific CD8⁺ T cells following administration of phosphatebuffered saline (PBS), recombinant IL-2 (rIL-2), or an IL-2/synTac ofthe present disclosure. The right panel depicts antigen-specific andnon-antigen-specific responses following administration of PBS, rIL-2,or an IL-2/synTac of the present disclosure.

FIG. 16A-16B depict dose escalation (FIG. 16A) and route ofadministration (FIG. 16B) effects.

FIG. 17A-17B depict the effect of IL-2 copy number on in vivo efficacyagainst a tumor.

FIG. 18 depicts the serum half-life of an IL-2/synTac of the presentdisclosure, following intraperitoneal administration of the IL-2/synTacin an amount of 10 mg/kg.

FIG. 19 depicts stability of an IL-2/synTac of the present disclosure 2hours following intraperitoneal administration of the IL-2/synTac in anamount of 10 mg/kg.

FIG. 20 depicts size exclusion chromatography data on an IL-2/synTac ofthe present disclosure after keeping the IL-2/synTac at 4° C. or 37° C.for 5 days.

FIG. 21 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, with a leader peptide, where theIL-2/synTac heavy chain comprises an IgG1 Fc with an N297A substitution.

FIG. 22 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, without a leader peptide, wherethe IL-2/synTac heavy chain comprises an IgG1 Fc with an N297Asubstitution.

FIG. 23A-23B provide a nucleotide sequence (FIG. 23A) encoding theIL-2/synTac heavy chain depicted in FIG. 21 ; and a key (FIG. 23B) tothe sequence.

FIG. 24 provides an amino acid sequence of a heavy chain of anIL-2/synTac, with a leader peptide, where the IL-2/synTac heavy chaincomprises an IgG1 Fc with L234A and L235A substitutions.

FIG. 25 provides an amino acid sequence of a heavy chain of anIL-2/synTac, without a leader peptide, where the IL-2/synTac heavy chaincomprises an IgG1 Fc with L234A and L235A substitutions.

FIG. 26A-26B provide a nucleotide sequence (FIG. 26A) encoding theIL-2/synTac heavy chain depicted in FIG. 24 ; and a key (FIG. 26B) tothe sequence.

FIG. 27 provides an amino acid sequence of a heavy chain of anIL-2/synTac, with a leader peptide, where the IL-2/synTac heavy chaincomprises an IgG1 Fc with L234F, L235E, and P331S substitutions.

FIG. 28 provides an amino acid sequence of a heavy chain of anIL-2/synTac, without a leader peptide, where the IL-2/synTac heavy chaincomprises an IgG1 Fc with L234F, L235E, and P331S substitutions.

FIG. 29A-29B provide a nucleotide sequence (FIG. 29A) encoding theIL-2/synTac heavy chain depicted in FIG. 27 ; and a key (FIG. 29B) tothe sequence.

FIG. 30 provides an amino acid sequence of a light chain of anIL-2/synTac, with a leader peptide, where the IL-2/synTac light chaincomprises a human papilloma virus (HPV) E7 epitope.

FIG. 31 provides an amino acid sequence of a light chain of anIL-2/synTac, without a leader peptide, where the IL-2/synTac light chaincomprises an HPV E7 epitope.

FIG. 32 provides a nucleotide sequence encoding the IL-2/synTac lightchain depicted in FIG. 30 .

FIG. 33A-33D provide amino acid sequences of a wild-type human IgG1 Fc(FIG. 33A), an IgG1 Fc with L234F, L235E, and P33IS substitutions (FIG.33B), an IgG1 Fc with an N297A substitution (FIG. 33C), and an IgG1 Fcwith L234A and L235A substitutions (FIG. 33D).

FIG. 34A-34C provide amino acid sequence of a β2-microglobulin (R12C)polypeptide (FIG. 34A), a variant IL-2 (H16A; F42A) polypeptide (FIG.34B), and a Class I MHC-H chain A0201 (Y84A; A236C) (FIG. 34C).

FIG. 35 depicts synergistic effects of an IL-2/synTac and an anti-PD1antibody on reducing tumor volume.

FIG. 36A-36IIII provide an amino acid sequence of a 4-1BBL (FIG. 36A)and examples of variant 4-1BBL polypeptides (FIG. 36B-36IIII).

FIG. 37 provides an amino acid sequence of 4-1BB.

FIG. 38A-38B depicts interferon-gamma (IFN-γ) secretion by target cellscontacted with a synTac polypeptide for 3 days (FIG. 38A) or 5 days(FIG. 8B) according to an embodiment of the present disclosure.

FIG. 39A-39B depicts interleukin-2 (IL-2) secretion by target cellscontacted with a synTac polypeptide for 3 days (FIG. 39A) or 5 days(FIG. 9B) according to an embodiment of the present disclosure.

FIG. 40A-40B depicts interleukin-6 (IL-6) secretion by target cellscontacted with a synTac polypeptide for 3 days (FIG. 40A) or 5 days(FIG. 40B) according to an embodiment of the present disclosure.

FIG. 41A-41B depicts tumor necrosis factor-alpha (TNFα) secretion bytarget cells contacted with a synTac polypeptide for 3 days (FIG. 41A)or 5 days (FIG. 41B) according to an embodiment of the presentdisclosure.

FIG. 42A-42B depicts interleukin-10 (IL-10) secretion by target cellscontacted with a synTac polypeptide for 3 days (FIG. 42A) or 5 days(FIG. 42B) according to an embodiment of the present disclosure.

FIG. 43A-43B depicts interleukin-17A (IL-17A) secretion by target cellscontacted with a synTac polypeptide for 3 days (FIG. 43A) or 5 days(FIG. 43B) according to an embodiment of the present disclosure.

FIG. 44A-44B depicts interleukin-4 (IL-4) secretion by target cellscontacted with a synTac polypeptide for 3 days (FIG. 44A) or 5 days(FIG. 44B) according to an embodiment of the present disclosure.

FIG. 45 depicts proliferation of target cells contacted with a synTacpolypeptide according to an embodiment of the present disclosure.

FIG. 46 depicts viability of target cells contacted with a synTacpolypeptide according to an embodiment of the present disclosure.

FIG. 47 depicts expression levels of various synTac polypeptidesproduced in CHO cells.

FIG. 48 depicts the in vivo effect of a synTac polypeptide of thepresent disclosure on tumor volume.

FIG. 49 depicts the effect of co-administration of various doses of a4-1BBL/synTac and an anti-PD1 antibody on tumor mass and percentgranzyme B⁺ tumor infiltrating lymphocytes (TILs).

FIG. 50A-50B provide amino acid sequences of PD-L1 polypeptides.

FIG. 51 provides an amino acid sequence of a CD80 polypeptide.

FIG. 52 provides an amino acid sequence of an ICOS-L polypeptide.

FIG. 53 provides an amino acid sequence of an OX40L polypeptide.

FIG. 54 provides an amino acid sequence of a PD-L2 polypeptide.

FIG. 55 provides an amino acid sequence of a CD86 (B7-2) polypeptide.

FIG. 56 provides an amino acid sequence of a Fas ligand (FAS-L)polypeptide.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequenceidentity can be determined in a number of different ways. To determinesequence identity, sequences can be aligned using various convenientmethods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT,etc.), available over the world wide web at sites includingncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/,ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See,e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide containingside chains consisting of asparagine and glutamine; a group of aminoacids having aromatic side chains consists of phenylalanine, tyrosine,and tryptophan; a group of amino acids having basic side chains consistsof lysine, arginine, and histidine; a group of amino acids having acidicside chains consists of glutamate and aspartate; and a group of aminoacids having sulfur containing side chains consists of cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine-glycine, and asparagine-glutamine.

“Binding” as used herein (e.g. with reference to binding of a T-cellmodulatory multimeric polypeptide to a polypeptide (e.g., a T-cellreceptor) on a T cell) refers to a non-covalent interaction between.Binding interactions are generally characterized by a dissociationconstant (K_(D)) of less than 10⁻⁶ M, less than 10⁻⁷ M, less than 10⁻⁸M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹ M, less than10⁻¹² M, less than 10⁻³ M, less than 10⁻¹⁴ M, or less than 10⁻¹⁵ M.“Affinity” refers to the strength of binding, increased binding affinitybeing correlated with a lower K_(D).

The term “immunological synapse” or “immune synapse” as used hereingenerally refers to the natural interface between two interacting immunecells of an adaptive immune response including, e.g., the interfacebetween an antigen-presenting cell (APC) or target cell and an effectorcell, e.g., a lymphocyte, an effector T cell, a natural killer cell, andthe like. An immunological synapse between an APC and a T cell isgenerally initiated by the interaction of a T cell antigen receptor andmajor histocompatibility complex molecules, e.g., as described inBromley et al., Annu Rev Immunol. 2001; 19:375-96; the disclosure ofwhich is incorporated herein by reference in its entirety.

“T cell” includes all types of immune cells expressing CD3, includingT-helper cells (CD4⁺ cells), cytotoxic T-cells (CD8⁺ cells),T-regulatory cells (Treg), and NK-T cells.

“Co-stimulatory polypeptide,” (also referred to herein as an“immunomodulatory polypeptide”) as the term is used herein, includes apolypeptide on an antigen presenting cell (APC) (e.g., a dendritic cell,a B cell, and the like) that specifically binds a cognate co-stimulatorypolypeptide (also referred to herein as a “cognate co-immunomodulatorypolypeptide”) on a T cell, thereby providing a signal which, in additionto the primary signal provided by, for instance, binding of a TCR/CD3complex with a major histocompatibility complex (MHC) polypeptide loadedwith peptide, mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. Aco-stimulatory ligand can include, but is not limited to, CD7, B7-1(CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL),inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule(ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxinbeta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody thatbinds Toll ligand receptor and a ligand that specifically binds withB7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibodythat specifically binds with a co-stimulatory molecule present on a Tcell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40,PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

A “modulatory domain” (“MOD”) of a T-cell modulatory multimericpolypeptide comprises a co-stimulatory polypeptide, e.g., an IL-2polypeptide, such as a variant IL-2 polypeptide.

“Heterologous,” as used herein, means a nucleotide or polypeptide thatis not found in the native nucleic acid or protein, respectively.

“Recombinant,” as used herein, means that a particular nucleic acid (DNAor RNA) is the product of various combinations of cloning, restriction,polymerase chain reaction (PCR) and/or ligation steps resulting in aconstruct having a structural coding or non-coding sequencedistinguishable from endogenous nucleic acids found in natural systems.DNA sequences encoding polypeptides can be assembled from cDNA fragmentsor from a series of synthetic oligonucleotides, to provide a syntheticnucleic acid which is capable of being expressed from a recombinanttranscriptional unit contained in a cell or in a cell-free transcriptionand translation system.

The terms “recombinant expression vector,” or “DNA construct” are usedinterchangeably herein to refer to a DNA molecule comprising a vectorand one insert. Recombinant expression vectors are usually generated forthe purpose of expressing and/or propagating the insert(s), or for theconstruction of other recombinant nucleotide sequences. The insert(s)may or may not be operably linked to a promoter sequence and may or maynot be operably linked to DNA regulatory sequences.

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies that retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies (scAb), single domain antibodies (dAb), singledomain heavy chain antibodies, a single domain light chain antibodies,bi-specific antibodies, multi-specific antibodies, and fusion proteinscomprising an antigen-binding (also referred to herein as antigenbinding) portion of an antibody and a non-antibody protein. Alsoencompassed by the term are Fab′, Fv, F(ab′)₂, and or other antibodyfragments that retain specific binding to antigen, and monoclonalantibodies. As used herein, a monoclonal antibody is an antibodyproduced by a group of identical cells, all of which were produced froma single cell by repetitive cellular replication. That is, the clone ofcells only produces a single antibody species. While a monoclonalantibody can be produced using hybridoma production technology, otherproduction methods known to those skilled in the art can also be used(e.g., antibodies derived from antibody phage display libraries). Anantibody can be monovalent or bivalent. An antibody can be an Igmonomer, which is a “Y-shaped” molecule that consists of fourpolypeptide chains: two heavy chains and two light chains connected bydisulfide bonds.

The term “humanized antibody” as used herein refers to an antibodycomprising portions of antibodies of different origin, wherein at leastone portion comprises amino acid sequences of human origin. For example,a humanized antibody can comprise portions derived from animmunoglobulin of nonhuman origin with the requisite specificity, suchas a mouse, and from immunoglobulin sequences of human origin (e.g.,chimeric immunoglobulin), joined together chemically by conventionaltechniques (e.g., synthetic) or prepared as a contiguous polypeptideusing genetic engineering techniques (e.g., DNA encoding the proteinportions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanized antibodyis an antibody containing one or more antibody chains comprising a CDRderived from an antibody of nonhuman origin and a framework regionderived from a light and/or heavy chain of human origin (e.g.,CDR-grafted antibodies with or without framework changes). Chimeric orCDR-grafted single chain antibodies are also encompassed by the termhumanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al., EuropeanPatent Application No. 0,519,596 A1. See also, Ladner et al., U.S. Pat.No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al.,Science, 242: 423-426 (1988)), regarding single chain antibodies.

For example, humanized antibodies can be produced using synthetic and/orrecombinant nucleic acids to prepare genes (e.g., cDNA) encoding thedesired humanized chain. For example, nucleic acid (e.g., DNA) sequencescoding for humanized variable regions can be constructed using PCRmutagenesis methods to alter DNA sequences encoding a human or humanizedchain, such as a DNA template from a previously humanized variableregion (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404(1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.For example, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003)Trends Biotechnol. 21:484); single-chain antibody molecules; andmulti-specific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH₁) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH₁ domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these classes can be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Thesubclasses can be further divided into types, e.g., IgG2a and IgG2b.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise theV_(H) and V_(L) domains of antibody, wherein these domains are presentin a single polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains, which enables the sFv to form the desired structure for antigenbinding. For a review of sFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents (e.g., an antibody and anantigen) and is expressed as a dissociation constant (K_(D)). Affinitycan be at least 1-fold greater, at least 2-fold greater, at least 3-foldgreater, at least 4-fold greater, at least 5-fold greater, at least6-fold greater, at least 7-fold greater, at least 8-fold greater, atleast 9-fold greater, at least 10-fold greater, at least 20-foldgreater, at least 30-fold greater, at least 40-fold greater, at least50-fold greater, at least 60-fold greater, at least 70-fold greater, atleast 80-fold greater, at least 90-fold greater, at least 100-foldgreater, or at least 1,000-fold greater, or more, than the affinity ofan antibody for unrelated amino acid sequences. Affinity of an antibodyto a target protein can be, for example, from about 100 nanomolar (nM)to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or fromabout 100 nM to about 1 femtomolar (fM) or more. As used herein, theterm “avidity” refers to the resistance of a complex of two or moreagents to dissociation after dilution. The terms “immunoreactive” and“preferentially binds” are used interchangeably herein with respect toantibodies and/or antigen-binding fragments.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. “Specific binding” refers to binding with anaffinity of at least about 10⁻⁷ M or greater, e.g., 5×10⁻⁷ M, 10⁻⁸ M,5×10⁻⁸ M, and greater. “Non-specific binding” refers to binding with anaffinity of less than about 10⁻⁷ M, e.g., binding with an affinity of10⁻⁶ M, 10⁻¹ M, 10⁻⁴ M, etc.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of proteins of immunological interest” (1991) (also referredto herein as Kabat 1991); by Chothia et al., J. Mol. Biol. 196:901-917(1987) (also referred to herein as Chothia 1987); and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or grafted antibodies or variants thereof is intended tobe within the scope of the term as defined and used herein. The aminoacid residues, which encompass the CDRs, as defined by each of the abovecited references are set forth in the table below as a comparison. TheCDRs listed in Table 2 were defined in accordance with Kabat 1991.

TABLE CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR-1 31-35 26-3230-35 V_(H) CDR-2 50-65 53-55 47-58 V_(H) CDR-3  95-102  96-101  93-101V_(L) CDR-1 24-34 26-32 30-36 V_(L) CDR-2 50-56 50-52 46-55 V_(L) CDR-389-97 91-96 89-96 ¹Residue numbering follows the nomenclature of Kabatet al., supra ²Residue numbering follows the nomenclature of Chothia etal., supra

As used herein, the terms “CDR-L1”, “CDR-L2”, and “CDR-L3” refer,respectively, to the first, second, and third CDRs in a light chainvariable region. As used herein, the terms “CDR-H1”, “CDR-H2”, and“CDR-H3” refer, respectively, to the first, second, and third CDRs in aheavy chain variable region. As used herein, the terms “CDR-1”, “CDR-2”,and “CDR-3” refer, respectively, to the first, second and third CDRs ofeither chain's variable region.

As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally a discontinuous amino acidsequence between about 100-120 amino acids in length but is intended toreference only those amino acids outside of the CDRs. As used herein,the term “framework region” is intended to mean each domain of theframework that is separated by the CDRs.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease or symptom in a mammal, and includes:(a) preventing the disease or symptom from occurring in a subject whichmay be predisposed to acquiring the disease or symptom but has not yetbeen diagnosed as having it; (b) inhibiting the disease or symptom,i.e., arresting its development; or (c) relieving the disease, i.e.,causing regression of the disease. The therapeutic agent may beadministered before, during or after the onset of disease or injury. Thetreatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, is of particularinterest. Such treatment is desirably performed prior to complete lossof function in the affected tissues. The subject therapy will desirablybe administered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired. Mammals include, e.g.,humans, non-human primates, rodents (e.g., rats; mice), lagomorphs(e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, andthe like), etc.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “avariant IL-2 polypeptide” includes a plurality of such polypeptides andreference to “the Class I HLA heavy chain polypeptide” includesreference to one or more Class I HLA heavy chain polypeptides andequivalents thereof known to those skilled in the art, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides treatment methods comprisingadministering to an individual in need thereof a T-cell modulatorymultimeric polypeptide (a “synTac” multimeric polypeptide) and at leastone additional therapeutic agent. In some cases, the at least oneadditional therapeutic agent is an immune checkpoint inhibitor. In somecases, the immune checkpoint inhibitor is an antibody specific for theimmune checkpoint. The present disclosure provides methods comprisingadministering a multimeric polypeptide (synTac) and an immune checkpointinhibitor to an individual. The present disclosure provides methodscomprising administering a multimeric polypeptide (synTac) to anindividual who is undergoing treatment with immune checkpoint inhibitor.

A “T-cell modulatory multimeric polypeptide” is also referred herein toas a “synTac polypeptide” or a “synTac multimeric polypeptide” or simply“synTac.” A synTac polypeptide comprises a modulatory domain. In somecases, the modulatory domain comprises a wild-type amino acid sequence,e.g., an amino acid sequence found in a naturally-occurring modulatorypolypeptide. In some cases, the modulatory domain is a variantmodulatory domain, where the variant modulatory domain exhibits reducedbinding affinity to an immunomodulatory polypeptide, compared to theaffinity of a wild-type modulatory domain for the immunomodulatorypolypeptide. A synTac polypeptide can modulate the activity of a targetT-cell. A synTac polypeptide comprising a variant modulatory domainprovides for enhanced target cell specificity.

In some cases, a treatment method of the present disclosure comprisesadministering to an individual in need thereof a synTac and an immunecheckpoint inhibitor. In some cases, the synTac and the immunecheckpoint inhibitor provide synergistic effects, compared to theeffect(s) of the synTac when administered alone (in monotherapy) or theimmune checkpoint inhibitor alone (in monotherapy).

The combination of a synTac and an immune checkpoint inhibitor is insome cases more effective than the additive effects of the synTacadministered as monotherapy or the immune checkpoint inhibitoradministered as monotherapy. For example, in some cases, a synergisticeffect of a synTac and an immune checkpoint inhibitor permits the use oflower dosages of the synTac or the immune checkpoint inhibitor and/orless frequent administration of the synTac or the immune checkpointinhibitor to an individual in need thereof. The ability to utilize lowerdosages of therapeutic agents (a synTac or an immune checkpointinhibitor) and/or to administer such agents less frequently can reducetoxicity or other adverse side effects that may be associated with theadministration of the therapeutic agent in monotherapy, without reducingthe efficacy of the therapeutic agent in a treatment. In addition, asynergistic effect of a synTac and an immune checkpoint inhibitor canresult in enhanced clinical benefit, compared to the clinical benefitobtained with synTac monotherapy or immune checkpoint inhibitormonotherapy. Examples of clinical benefit include, e.g., reduced tumormass in an individual; reduced number of cancer cells in an individual;increased survival time of the individual; increased remission time; andthe like. Finally, a synergistic effect of a synTac and an immunecheckpoint inhibitor can be reduced adverse or unwanted side effectsassociated with synTac monotherapy or immune checkpoint inhibitormonotherapy.

Immune Checkpoint Inhibitors

Exemplary immune checkpoint inhibitors include inhibitors that targetimmune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96,CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM,arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA,CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. Insome cases, the immune checkpoint polypeptide is a stimulatorycheckpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR,CD122 and CD137. In some cases, the immune checkpoint polypeptide is aninhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA,CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA.

In some cases, the immune checkpoint inhibitor is an antibody specificfor an immune checkpoint. In some cases, the anti-immune checkpointantibody is a monoclonal antibody. In some cases, the anti-immunecheckpoint antibody is humanized, or de-immunized such that the antibodydoes not substantially elicit an immune response in a human. In somecases, the anti-immune checkpoint antibody is a humanized monoclonalantibody. In some cases, the anti-immune checkpoint antibody is ade-immunized monoclonal antibody. In some cases, the anti-immunecheckpoint antibody is a fully human monoclonal antibody. In some cases,the anti-immune checkpoint antibody inhibits binding of the immunecheckpoint polypeptide to a ligand for the immune checkpointpolypeptide. In some cases, the anti-immune checkpoint antibody inhibitsbinding of the immune checkpoint polypeptide to a receptor for theimmune checkpoint polypeptide.

Antibodies, e.g., monoclonal antibodies, that are specific for immunecheckpoints and that function as immune checkpoint inhibitors, are knownin the art. See, e.g., Wurz et al. (2016) Ther. Adv. Med. Oncol. 8:4;and Naidoo et al. (2015) Ann. Oncol. 26:2375.

Suitable anti-immune checkpoint antibodies include, but are not limitedto, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab(Curetech), AMP-224 (GlaxoSmithKline/Amplimmune), MPDL3280A (Roche),MDX-1105 (Medarex, Inc./Bristol Myer Squibb), MEDI-4736(Medimmune/AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY,(Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech,Ltd.), IMP321 (Immutep S.A.), MGA271 (Macrogenics), BMS-986016(Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab(Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (InnatePharma/Bristol-Myers Squibb), MEDI-6469 (Medlmmune/AZ), CP-870,893(Genentech), Mogamulizumab (Kyowa Hakko Kirin), Varlilumab (CellDexTherapeutics), Avelumab (EMD Serono), Galiximab (Biogen Idec), AMP-514(Amplimmune/AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLinkGenetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte); KN035; andcombinations thereof.

Suitable anti-LAG3 antibodies include, e.g., BMS-986016 and LAG525.Suitable anti-GITR antibodies include, e.g., TRX518, MK-4166,INCAGN01876, and MK-1248. Suitable anti-OX40 antibodies include, e.g.,MED10562, INCAGN01949, GSK2831781, GSK-3174998, MOXR-0916, PF-04518600,and LAG525. Suitable anti-VISTA antibodies are provided in, e.g., WO2015/097536.

A suitable dosage of an anti-immune checkpoint antibody is from about 1mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about1200 mg/kg per day, including from about 50 mg/kg to about 1200 mg/kgper day. Other representative dosages of such agents include about 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg,400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200mg/kg, and 2300 mg/kg per day. The effective dose of the antibody may beadministered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.

Anti-PD-1 Antibodies

In some cases, an immune checkpoint inhibitor is an anti-PD-1 antibody.

Suitable anti-PD-1 antibodies include, e.g., nivolumab, pembrolizumab(also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224. Insome cases, the anti-PD-1 monoclonal antibody is nivolumab,pembrolizumab or PDR001. Suitable anti-PD1 antibodies are described inU.S. Patent Publication No. 2017/0044259. For pidilizumab, see, e.g.,Rosenblatt et al. (2011) J. Immunother. 34:409-18.

In some cases, the anti-PD1 antibody is pembrolizumab. The amino acidsequence of the heavy chain of pembrolizumab is:

(SEQ ID NO: 51) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK.The amino acid sequence of the heavy chain variable (VH) region isunderlined.

The amino acid sequence of the light chain of pembrolizumab is:

(SEQ ID NO: 52) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.The amino acid sequence of the light chain variable (VL) region isunderlined.

In some cases, the anti-PD-1 antibody comprises the VH and VL regions ofpembrolizumab. In some cases, the anti-PD-1 antibody comprises heavy andlight chain CDRs of pembrolizumab.

In some cases, the anti-PD-1 antibody is nivolumab (also known asMDX-1106 or BMS-936558; see, e.g., Topalian et al. (2012) N. Eng. J.Med. 366:2443-2454; and U.S. Pat. No. 8,008,449). The amino acidsequence of the heavy chain of nivolumab is:

(SEQ ID NO: 53) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The amino acid sequence of the light chain of nivolumab is:

(SEQ ID NO: 54) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In some cases, the anti-PD-1 antibody comprises heavy and light chainCDRs of nivolumab.

Anti-CTLA4 Antibodies

In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab.For tremelimumab, see, e.g., Ribas et al. (2013) J. Clin. Oncol.31:616-22.

In some cases, the anti-CTLA-4 antibody is ipilimumab. The amino acidsequence of the heavy chain of ipilimumab is:

(SEQ ID NO: 55) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.The amino acid sequence of the VH region is underlined.

The amino acid sequence of the light chain of ipilimumab is:

(SEQ ID NO: 56) EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.The amino acid sequence of the VL region is underlined.

In some cases, the anti-CTLA4 antibody comprises the VH and VL regionsof ipilimumab. In some cases, the anti-CTLA4 antibody comprises heavyand light chain CDRs of ipilimumab.

Anti-PD-L1 Antibodies

In some cases, the immune checkpoint inhibitor is an anti-PD-L1monoclonal antibody. In some cases, the anti-PD-L1 monoclonal antibodyis BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, orMSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody isMPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see,e.g., WO 2011/066389. For atezolizumab, see, e.g., U.S. Pat. No.8,217,149.

In some cases, the anti-PD-L1 antibody is atezolizumab. The amino acidsequence of the heavy chain of atezolizumab is:

(SEQ ID NO: 57) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.

The amino acid sequence of the light chain of atezolizumab is:

(SEQ ID NO: 58) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In some cases, the anti-PD-L1 antibody comprises heavy and light chainCDRs of atezolizumab.

In some cases, the anti-PDL1 antibody is KN035, a fully humanizedanti-PD-L1 single domain antibody fused to a human IgG1 Fc polypeptide.Zhang et al. (2017) Cell Discov. 3:17004; and WO 2017/020801. Thesingle-domain antibody portion of KN035 can comprise the amino acidsequence:

(SEQ ID NO: 216) QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVS,where the underlined amino acids are CDR1, CDR2, and CDR3.T-Cell Modulatory Multimeric Polypeptides (synTacs)

Multimeric (e.g., heterodimeric, heterotrimeric) polypeptides suitablefor use in a method of the present disclosure are described below. Themultimeric polypeptides are T cell modulatory polypeptides, and are alsoreferred to herein as “T-cell modulatory multimeric polypeptides,” or“synTac” (for “immunological synapse for T cell activation”).

A T-cell modulatory multimeric polypeptide comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; ii) a first major histocompatibility complex (MHC) polypeptide;and b) a second polypeptide comprising, in order from N-terminus toC-terminus: i) a second MHC polypeptide; and ii) optionally animmunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, where themultimeric polypeptide comprises one or more immunomodulatory (“MOD”)domains, wherein the one or more immunomodulatory domain is: A) at theC-terminus of the first polypeptide; B) at the N-terminus of the secondpolypeptide; C) at the C-terminus of the second polypeptide; or D) atthe C-terminus of the first polypeptide and at the N-terminus of thesecond polypeptide. In some cases, a T-cell multimeric polypeptidecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; ii) a first MHC polypeptide; and iii) animmunomodulatory domain; and b) a second polypeptide comprising, inorder from N-terminus to C-terminus: i) a second MHC polypeptide; andii) an Ig Fc polypeptide. In some cases, a T-cell multimeric polypeptidecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) asecond polypeptide comprising, in order from N-terminus to C-terminus:i) an immunomodulatory domain; iii) a second MHC polypeptide; and ii) anIg Fc polypeptide. In some cases, a T-cell multimeric polypeptidecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) asecond polypeptide comprising, in order from N-terminus to C-terminus:i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) animmunomodulatory (“MOD”) domain. In some cases, a T-cell multimericpolypeptide comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus: i) an epitope; and ii) a first MHCpolypeptide; and b) a second polypeptide comprising, in order fromN-terminus to C-terminus: i) a second MHC polypeptide; and ii) animmunomodulatory domain. In some cases, a T-cell multimeric polypeptidecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) asecond polypeptide comprising, in order from N-terminus to C-terminus:i) an immunomodulatory domain; and ii) a second MHC polypeptide. In somecases, a T-cell multimeric polypeptide comprises: a) a first polypeptidecomprising, in order from N-terminus to C-terminus: i) an epitope; ii) afirst MHC polypeptide; and iii) an immunomodulatory domain; and b) asecond polypeptide comprising, in order from N-terminus to C-terminus:i) a second MHC polypeptide.

In some cases, a multimeric polypeptide comprises a non-Ig scaffold. Forexample, in some cases, the non-Ig scaffold is an XTEN polypeptide, atransferrin polypeptide, an Fc receptor polypeptide, an elastin-likepolypeptide, a silk-like polypeptide, or a silk-elastin-likepolypeptide.

In some cases, the first MHC polypeptide is a 02-microglobulin (β2M)polypeptide; and the second MHC polypeptide is an MHC class I heavychain polypeptide. A suitable 32-M polypeptide comprises an amino acidsequence having at least 85%, at least 90%, at least 95%, at least 98%,at least 99%, or 100%, amino acid sequence identity to the amino acidsequence of a β2M polypeptide depicted in FIG. 6 . In some cases, theMHC class I heavy chain polypeptide is an HLA-A, an HLA-B, or an HLA-Cheavy chain. In some cases, the MHC class I heavy chain polypeptidecomprises an amino acid sequence having at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to the amino acid sequence set forth in one of FIG. 5A-5C. Insome cases, the first MHC polypeptide is an MHC Class II alpha chainpolypeptide; and the second MHC polypeptide is an MHC class II betachain polypeptide.

The epitope present in a multimeric polypeptide can be a T-cell epitope.

In some cases, a multimeric polypeptide comprises an Ig Fc polypeptide.In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgAFc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fcpolypeptide comprises an amino acid sequence having at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to an amino acid sequence depicted in FIG. 4A-4C.

The first polypeptide and the second polypeptide of a multimericpolypeptide can be non-covalently associated. The first polypeptide andthe second polypeptide of a multimeric polypeptide can be covalentlylinked. The first polypeptide and the second polypeptide of a multimericpolypeptide can be covalently linked, where the covalent linkage is viaa disulfide bond. In some cases, the first MHC polypeptide or a linkerbetween the epitope and the first MHC polypeptide comprises an aminoacid substitution to provide a first Cys residue, and the second MHCpolypeptide comprises an amino acid substitution to provide a second Cysresidue, and wherein the disulfide linkage is between the first and thesecond Cys residues.

A multimeric polypeptide can include a linker between one or more of:the epitope and the first MHC polypeptide; two copies of theimmunomodulatory (“MOD”) polypeptide; the immunomodulatory polypeptideand the second MHC polypeptide; and the second MHC polypeptide and theIg Fc polypeptide.

Immunomodulatory polypeptides suitable for inclusion in a T-cellmultimeric polypeptide include, but are not limited to, a 4-1BBLpolypeptide, a B7-1 polypeptide; a B7-2 polypeptide, an ICOS-Lpolypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86polypeptide, an IL-2 polypeptide, a PD-L1 polypeptide, a FasLpolypeptide, and a PD-L2 polypeptide.

A multimeric polypeptide can include 2 or more immunomodulatorypolypeptides. A multimeric polypeptide can include 2 immunomodulatorypolypeptides. In some cases, the 2 immunomodulatory polypeptides are intandem. A multimeric polypeptide can include 3 immunomodulatorypolypeptides. In some cases, the 3 immunomodulatory polypeptides are intandem.

A multimeric polypeptide can comprise a third polypeptide, where thethird polypeptide comprises an immunomodulatory polypeptide comprisingan amino acid sequence having at least 90%, amino acid sequence identityto the immunomodulatory polypeptide of the first polypeptide or thesecond polypeptide. In some cases, the third polypeptide is covalentlylinked to the first polypeptide.

Examples of suitable multimeric polypeptides are described in WO2017/151940; WO 2017/201210; and PCT/US2017/067663. The disclosures ofWO 2017/151940, WO 2017/201210, and PCT/US2017/067663 are incorporatedby reference herein.

MHC Polypeptides

As noted above, a multimeric polypeptide of the present disclosureincludes MHC polypeptides. For the purposes of the instant disclosure,the term “major histocompatibility complex (MHC) polypeptides” is meantto include MHC polypeptides of various species, including human MHC(also referred to as human leukocyte antigen (HLA)) polypeptides, rodent(e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of othermammalian species (e.g., lagomorphs, non-human primates, canines,felines, ungulates (e.g., equines, bovines, ovines, caprines, etc.), andthe like. The term “MHC polypeptide” is meant to include Class I MHCpolypeptides (e.g., 3-2 microglobulin and MHC class I heavy chain) andMHC Class II polypeptides (e.g., MHC Class II α polypeptide and MHCClass II β polypeptide).

As noted above, in some embodiments of a multimeric polypeptide of thepresent disclosure, the first and the second MHC polypeptides are ClassI MHC polypeptides; e.g., in some cases, the first MHC polypeptide is anMHC Class I 02-microglobulin (β2M) polypeptide, and the second MHCpolypeptide is an MHC Class I heavy chain (H chain). In other cases, thefirst and the second MHC polypeptides are Class II MHC polypeptides;e.g., in some cases, the first MHC polypeptide is an MHC Class IIα-chain polypeptide, and the second MHC polypeptide is an MHC Class IIR-chain polypeptide. In other cases, the first polypeptide is an MHCClass II R-chain polypeptide, and the second MHC polypeptide is an MHCClass II α-chain polypeptide.

In some cases, an MHC polypeptide of a multimeric polypeptide of thepresent disclosure is a human MHC polypeptide, where human MHCpolypeptides are also referred to as “human leukocyte antigen” (“HLA”)polypeptides. In some cases, an MHC polypeptide of a multimericpolypeptide of the present disclosure is a Class I HLA polypeptide,e.g., a 02-microglobulin polypeptide, or a Class I HLA heavy chainpolypeptide. Class I HLA heavy chain polypeptides include HLA-A heavychain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chainpolypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chainpolypeptides, and HLA-G heavy chain polypeptides. In some cases, an MHCpolypeptide of a multimeric polypeptide of the present disclosure is aClass II HLA polypeptide, e.g., a Class II HLA α chain or a Class II HLAβ chain. MHC Class II polypeptides include MCH Class II DP a and βpolypeptides, DM α and β polypeptides, DOA α and β polypeptides, DOB αand β polypeptides, DQ α and βpolypeptides, and DR α and β polypeptides.

In some cases, an MHC Class I heavy chain polypeptide of a multimericpolypeptide can comprise an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to an amino acidsequence depicted in one of FIG. 5A-5C.

HLA-A

As an example, an MHC Class I heavy chain polypeptide of a multimericpolypeptide can comprise an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following humanHLA-A heavy chain amino acid sequence:

(SEQ ID NO: 59) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP.

HLA-A (Y84A; A236C)

In some cases, the MHC Class I heavy chain polypeptide comprises Y84Aand A236C substitutions. For example, in some cases, the MHC Class Iheavy chain polypeptide comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-A heavy chain (Y84A; A236C) amino acid sequence:GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:50), whereamino acid 84 is Ala and amino acid 236 is Cys. In some cases, theCys-236 forms an interchain disulfide bond with Cys-12 of a variant β2Mpolypeptide that comprises an R12C substitution.

HLA-A (Y84C; A139C)

In some cases, the MHC Class I heavy chain polypeptide comprises Y84Cand A139C substitutions. For example, in some cases, the MHC Class Iheavy chain polypeptide comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-A heavy chain (Y84C; A139C) amino acid sequence:GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:196), whereamino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84forms an intrachain disulfide bond with Cys-139.

HLA-A A11 (HLA-A11)

As one non-limiting example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-A All (also referred to as “HLA-A11”) heavy chainamino acid sequence:

(SEQ ID NO: 197) GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE.Such an MHC Class I heavy chain may be prominent in Asian populations,including populations of individuals of Asian descent.

HLA-A A11 (Y84A; A236C)

As one non-limiting example, in some cases, the MHC Class I heavy chainpolypeptide is an HLA-A All allele that comprises Y84A and A236Csubstitutions. For example, in some cases, the MHC Class I heavy chainpolypeptide comprises an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following humanHLA-A All heavy chain (Y84A; A236C) amino acid sequence:

(SEQ ID NO: 198) GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRG A YNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE,where amino acid 84 is Ala and amino acid 236 is Cys. In some cases, theCys-236 forms an interchain disulfide bond with Cys-12 of a variant β2Mpolypeptide that comprises an R12C substitution.

HLA-B

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-B heavy chain amino acid sequence:

(SEQ ID NO: 199) GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP.

HLA-B (Y84A; A236C)

As one non-limiting example, in some cases, the MHC Class I heavy chainpolypeptide is an HLA-B polypeptide that comprises Y84A and A236Csubstitutions. For example, in some cases, the MHC Class I heavy chainpolypeptide comprises an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following humanHLA-B heavy chain (Y84A; A236C) amino acid sequence:

(SEQ ID NO: 200) GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRG A YNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP,where amino acid 84 is Ala and amino acid 236 is Cys. In some cases, theCys-236 forms an interchain disulfide bond with Cys-12 of a variant β2Mpolypeptide that comprises an R12C substitution.

HLA-B (Y84C; A139C)

In some cases, the MHC Class I heavy chain polypeptide comprises Y84Cand A139C substitutions. For example, in some cases, the MHC Class Iheavy chain polypeptide comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-B heavy chain (Y84C; A139C) amino acid sequence:

(SEQ ID NO: 201) GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRG C YNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADT C AQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP,where amino acid 84 is Cys and amino acid 139 is Cys. In some cases,Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-C

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-C heavy chain amino acid sequence:

(SEQ ID NO: 202) CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP.

HLA-C(Y84A; A236C)

As one non-limiting example, in some cases, the MHC Class I heavy chainpolypeptide is an HLA-C polypeptide that comprises Y84A and A236Csubstitutions. For example, in some cases, the MHC Class I heavy chainpolypeptide comprises an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to the following humanHLA-C heavy chain (Y84A; A236C) amino acid sequence:

(SEQ ID NO: 203) CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRG A YNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP,where amino acid 84 is Ala and amino acid 236 is Cys. In some cases, theCys-236 forms an interchain disulfide bond with Cys-12 of a variant β2Mpolypeptide that comprises an R12C substitution.

HLA-C(Y84C; A139C)

In some cases, the MHC Class I heavy chain polypeptide comprises Y84Cand A139C substitutions. For example, in some cases, the MHC Class Iheavy chain polypeptide comprises an amino acid sequence having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99%, or 100%, amino acid sequence identity to thefollowing human HLA-C heavy chain (Y84C; A139C) amino acid sequence:

(SEQ ID NO: 204) CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRG C YNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADT C AQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP,where amino acid 84 is Cys and amino acid 139 is Cys. In some cases,Cys-84 forms an intrachain disulfide bond with Cys-139.

In some cases, an MHC Class I heavy chain polypeptide of a multimericpolypeptide can comprise an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to an amino acidsequence depicted in one of FIG. 3A-3C.

As an example, an MHC Class I heavy chain polypeptide of a multimericpolypeptide can comprise an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 25-365of the amino acid sequence of the human HLA-A heavy chain polypeptidedepicted in FIG. 3A.

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 25-362 of the amino acid sequence of the human HLA-B heavy chainpolypeptide depicted in FIG. 3B.

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to aminoacids 25-362 of the amino acid sequence of the human HLA-C heavy chainpolypeptide depicted in FIG. 3C.

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide can comprise an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 60) GPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQEGPEYWEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLLRGYQQFAYDGRDYIALNEDLKTWTAADTAALITRRKWEQAGDAEYYRAYLEGECVEWLRRYLELGNETLLRTDSPKAHVTYHPRSQVDVTLRCWALGFYPADITLTWQLNGEDLTQDMELVETRPAGDGTFQKWAAVVVPLGKEQNYTCHVHHKGLPEPLTLRW.

A β2-microglobulin (β2M) polypeptide of a multimeric polypeptide can bea human β2M polypeptide, a non-human primate β2M polypeptide, a murineβ2M polypeptide, and the like. In some instances, a β2M polypeptidecomprises an amino acid sequence having at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or100%, amino acid sequence identity to a β2M amino acid sequence depictedin FIG. 6 . In some instances, a β2M polypeptide comprises an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to amino acids 21 to 119 of a β2M amino acid sequence depictedin FIG. 6 .

In some cases, an MHC polypeptide comprises a single amino acidsubstitution relative to a reference MHC polypeptide (where a referenceMHC polypeptide can be a wild-type MHC polypeptide), where the singleamino acid substitution substitutes an amino acid with a cysteine (Cys)residue. Such cysteine residues, when present in an MHC polypeptide of afirst polypeptide of a multimeric polypeptide of the present disclosure,can form a disulfide bond with a cysteine residue present in a secondpolypeptide chain of a multimeric polypeptide of the present disclosure.

In some cases, a first MHC polypeptide in a first polypeptide of amultimeric polypeptide, and/or the second MHC polypeptide in the secondpolypeptide of a multimeric polypeptide, includes an amino acidsubstitution to substitute an amino acid with a cysteine, where thesubstituted cysteine in the first MHC polypeptide forms a disulfide bondwith a cysteine in the second MHC polypeptide, where a cysteine in thefirst MHC polypeptide forms a disulfide bond with the substitutedcysteine in the second MHC polypeptide, or where the substitutedcysteine in the first MHC polypeptide forms a disulfide bond with thesubstituted cysteine in the second MHC polypeptide.

For example, in some cases, one of following pairs of residues in an HLAβ2-microglobulin and an HLA Class I heavy chain is substituted withcysteines (where residue numbers are those of the maturepolypeptide): 1) β2M residue 12, HLA Class I heavy chain residue 236; 2)β2M residue 12, HLA Class I heavy chain residue 237; 3) β2M residue 8,HLA Class I heavy chain residue 234; 4) β2M residue 10, HLA Class Iheavy chain residue 235; 5) β2M residue 24, HLA Class I heavy chainresidue 236; 6) β2M residue 28, HLA Class I heavy chain residue 232; 7)β2M residue 98, HLA Class I heavy chain residue 192; 8) β2M residue 99,HLA Class I heavy chain residue 234; 9) β2M residue 3, HLA Class I heavychain residue 120; 10) β2M residue 31, HLA Class I heavy chain residue96; 11) β2M residue 53, HLA Class I heavy chain residue 35; 12) β2Mresidue 60, HLA Class I heavy chain residue 96; 13) β2M residue 60, HLAClass I heavy chain residue 122; 14) β2M residue 63, HLA Class I heavychain residue 27; 15) β2M residue Arg3, HLA Class I heavy chain residueGly120; 16) β2M residue His31, HLA Class I heavy chain residue Gln96;17) β2M residue Asp53, HLA Class I heavy chain residue Arg35; 18) β2Mresidue Trp60, HLA Class I heavy chain residue Gln96; 19) β2M residueTrp60, HLA Class I heavy chain residue Asp122; 20) β2M residue Tyr63,HLA Class I heavy chain residue Tyr27; 21) β2M residue Lys6, HLA Class Iheavy chain residue Glu232; 22) β2M residue Gln8, HLA Class I heavychain residue Arg234; 23) β2M residue Tyr10, HLA Class I heavy chainresidue Pro235; 24) β2M residue Ser11, HLA Class I heavy chain residueGln242; 25) β2M residue Asn24, HLA Class I heavy chain residue Ala236;26) β2M residue Ser28, HLA Class I heavy chain residue Glu232; 27) β2Mresidue Asp98, HLA Class I heavy chain residue His192; and 28) β2Mresidue Met99, HLA Class I heavy chain residue Arg234. The amino acidnumbering of the MHC/HLA Class I heavy chain is in reference to themature MHC/HLA Class I heavy chain, without a signal peptide. Forexample, in the amino acid sequence depicted in FIG. 5A, which includesa signal peptide, Gly120 is Gly144; Gln96 is Gln120; etc. In some cases,the β2M polypeptide comprises an R12C substitution, and the HLA Class Iheavy chain comprises an A236C substitution; in such cases, a disulfidebond forms between Cys-12 of the β2M polypeptide and Cys-236 of the HLAClass I heavy chain. For example, in some cases, residue 236 of themature HLA-A amino acid sequence (i.e., residue 260 of the amino acidsequence depicted in FIG. 5A) is substituted with a Cys. In some cases,residue 236 of the mature HLA-B amino acid sequence (i.e., residue 260of the amino acid sequence depicted in FIG. 5B) is substituted with aCys. In some cases, residue 236 of the mature HLA-C amino acid sequence(i.e., residue 260 of the amino acid sequence depicted in FIG. 5C) issubstituted with a Cys. In some cases, residue 32 (corresponding toArg-12 of mature β2M) of an amino acid sequence depicted in FIG. 6 issubstituted with a Cys.

In some cases, a β2M polypeptide comprises the amino acid sequence:IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:61). In somecases, a β2M polypeptide comprises the amino acid sequence: IQRTPKIQVYSCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEFTPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:48).

In some cases, an HLA Class I heavy chain polypeptide comprises theamino acid sequence:

(SEQ ID NO: 59) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP.

In some cases, an HLA Class I heavy chain polypeptide comprises theamino acid sequence:

(SEQ ID NO: 62) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP.

In some cases, an HLA Class I heavy chain polypeptide comprises theamino acid sequence:

(SEQ ID NO: 50) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE

In some cases, the β2M polypeptide comprises the following amino acidsequence:

IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:48); and the HLAClassI heavy chain polypeptide of a multimeric polypeptide of thepresent disclosure comprises the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:62),where the Cys residues that are underlined and in bold form a disulfidebond with one another in the multimeric polypeptide.

In some cases, the β2M polypeptide comprises the amino acid sequence:IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTFTPTEKDEYACRVNHVTLSQPKTVKWDRDM (SEQ ID NO:48).

Scaffold Polypeptides

A T-cell modulatory multimeric polypeptide comprises an Fc polypeptide,or another suitable scaffold polypeptide.

Suitable scaffold polypeptides include antibody-based scaffoldpolypeptides and non-antibody-based scaffolds. Non-antibody-basedscaffolds include, e.g., albumin, an XTEN (extended recombinant)polypeptide, transferrin, an Fc receptor polypeptide, an elastin-likepolypeptide (see, e.g., Hassouneh et al. (2012) Methods Enzymol.502:215; e.g., a polypeptide comprising a pentapeptide repeat unit of(Val-Pro-Gly-X-Gly; SEQ ID NO:212), where X is any amino acid other thanproline), an albumin-binding polypeptide, a silk-like polypeptide (see,e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci.357:165), a silk-elastin-like polypeptide (SELP; see, e.g., Megeed etal. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTENpolypeptides include, e.g., those disclosed in WO 2009/023270, WO2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582; seealso Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitablealbumin polypeptides include, e.g., human serum albumin.

Suitable scaffold polypeptides will in some cases be a half-lifeextending polypeptides. Thus, in some cases, a suitable scaffoldpolypeptide increases the in vivo half-life (e.g., the serum half-life)of the multimeric polypeptide, compared to a control multimericpolypeptide lacking the scaffold polypeptide. For example, in somecases, a scaffold polypeptide increases the in vivo half-life (e.g., theserum half-life) of the multimeric polypeptide, compared to a controlmultimeric polypeptide lacking the scaffold polypeptide, by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 50%, at least about 2-fold, at least about 2.5-fold, atleast about 5-fold, at least about 10-fold, at least about 25-fold, atleast about 50-fold, at least about 100-fold, or more than 100-fold. Asan example, in some cases, an Fc polypeptide increases the in vivohalf-life (e.g., the serum half-life) of the multimeric polypeptide,compared to a control multimeric polypeptide lacking the Fc polypeptide,by at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 50%, at least about 2-fold, at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about25-fold, at least about 50-fold, at least about 100-fold, or more than100-fold.

Fc Polypeptides

In some cases, the first and/or the second polypeptide chain of amultimeric polypeptide comprises an Fc polypeptide. The Fc polypeptideof a multimeric polypeptide can be a human IgG1 Fc, a human IgG2 Fc, ahuman IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc polypeptidecomprises an amino acid sequence having at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to an amino acid sequence of an Fc regiondepicted in FIGS. 4A-C. In some cases, the Fc region comprises an aminoacid sequence having at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the human IgG1 Fc polypeptide depicted in FIG. 4A. In somecases, the Fc region comprises an amino acid sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the human IgG1 Fcpolypeptide depicted in FIG. 4A; and comprises a substitution of N77;e.g., the Fc polypeptide comprises an N77A substitution. In some cases,the Fc polypeptide comprises an amino acid sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the human IgG2 Fcpolypeptide depicted in FIG. 4A; e.g., the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 99-325 of the human IgG2 Fc polypeptidedepicted in FIG. 4A. In some cases, the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the human IgG3 Fc polypeptide depicted in FIG. 4A;e.g., the Fc polypeptide comprises an amino acid sequence having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids19-246 of the human IgG3 Fc polypeptide depicted in FIG. 4A. In somecases, the Fc polypeptide comprises an amino acid sequence having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to the human IgMFc polypeptide depicted in FIG. 4B; e.g., the Fc polypeptide comprisesan amino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 1-276 to the human IgM Fc polypeptidedepicted in FIG. 4B. In some cases, the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the human IgA Fc polypeptide depicted in FIG. 4C;e.g., the Fc polypeptide comprises an amino acid sequence having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1-234 to the human IgA Fc polypeptide depicted in FIG. 4C.

In some cases, the Fc polypeptide present in a multimeric polypeptidecomprises the amino acid sequence depicted in FIG. 33A (human IgG1 Fc).In some cases, the Fc polypeptide present in a multimeric polypeptide ofthe present disclosure comprises the amino acid sequence depicted inFIG. 33A (human IgG1 Fc), except for a substitution of N297 with anamino acid other than asparagine. In some cases, the Fc polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence depicted in FIG. 33C (human IgG1 Fc comprisingan N297A substitution). In some cases, the Fc polypeptide present in amultimeric polypeptide of the present disclosure comprises the aminoacid sequence depicted in FIG. 33A (human IgG1 Fc), except for asubstitution of L234 with an amino acid other than leucine. In somecases, the Fc polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence depicted in FIG.33A (human IgG1 Fc), except for a substitution of L235 with an aminoacid other than leucine. In some cases, the Fc polypeptide present in amultimeric polypeptide of the present disclosure comprises the aminoacid sequence depicted in FIG. 33D (human IgG1 Fc comprising an L234Asubstitution and an L235A substitution). In some cases, the Fcpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence depicted in FIG. 33A (humanIgG1 Fc), except for a substitution of P331 with an amino acid otherthan proline; in some cases, the substitution is a P331S substitution.In some cases, the Fc polypeptide present in a multimeric polypeptide ofthe present disclosure comprises the amino acid sequence depicted inFIG. 33A (human IgG1 Fc), except for substitutions at L234 and L235 withamino acids other than leucine. In some cases, the Fc polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except forsubstitutions at L234 and L235 with amino acids other than leucine, anda substitution of P331 with an amino acid other than proline. In somecases, the Fc polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence depicted in FIG.33B (human IgG1 Fc comprising L234F, L235E, and P331S substitutions). Insome cases, the Fc polypeptide present in a multimeric polypeptide is anIgG1 Fc polypeptide that comprises L234A and L235A substitutions.

Linkers

A multimeric polypeptide can include linker peptides interposed between,e.g., an epitope and an MHC polypeptide; between an MHC polypeptide andan immunomodulatory polypeptide; between an MHC polypeptide and an Ig Fcpolypeptide; between a first immunomodulatory polypeptide and a secondimmunomodulatory polypeptide; or a between a second immunomodulatorypolypeptide and a third immunomodulatory polypeptide.

For example, a multimeric polypeptide can include linker peptidesinterposed between, e.g., an epitope and an MHC polypeptide; between anMHC polypeptide and an immunomodulatory polypeptide; between an MHCpolypeptide and an Ig Fc polypeptide; between a first variant IL-2polypeptide and a second variant IL-2 polypeptide; or a between a secondvariant IL-2 polypeptide and a third variant IL-2 polypeptide. Asanother example, a multimeric polypeptide can include linker peptidesinterposed between, e.g., an epitope and an MHC polypeptide; between anMHC polypeptide and an immunomodulatory polypeptide; between an MHCpolypeptide and an Ig Fc polypeptide; between a first variant 4-1BBLpolypeptide and a second variant 4-1BBL polypeptide; or a between asecond variant 4-1BBL polypeptide and a third variant 4-1BBLpolypeptide.

Suitable linkers (also referred to as “spacers”) can be readily selectedand can be of any of a number of suitable lengths, such as from 1 aminoacid to 25 amino acids, from 3 amino acids to 20 amino acids, from 2amino acids to 15 amino acids, from 3 amino acids to 12 amino acids,including 4 amino acids to 10 amino acids, 5 amino acids to 9 aminoacids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 aminoacids. A suitable linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids inlength.

Exemplary linkers include glycine polymers (G)_(n), glycine-serinepolymers (including, for example, (GS)_(n), (GSGGS)_(n) (SEQ ID NO:210)and (GGGS)_(n) (SEQ ID NO:211), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers can beused; both Gly and Ser are relatively unstructured, and therefore canserve as a neutral tether between components. Glycine polymers can beused; glycine accesses significantly more phi-psi space than evenalanine, and is much less restricted than residues with longer sidechains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).

Exemplary linkers can comprise amino acid sequences including, but notlimited to, GGSG (SEQ ID NO:65), GGSGG (SEQ ID NO:66), GSGSG (SEQ IDNO:67), GSGGG (SEQ ID NO:68), GGGSG (SEQ ID NO:69), GSSSG (SEQ IDNO:70), and the like. Exemplary linkers can include, e.g., Gly(Ser₄)n,where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, a linkercomprises the amino acid sequence (GSSSS)n (SEQ ID NO:71), where n is 4.In some cases, a linker comprises the amino acid sequence (GSSSS)n (SEQID NO:72), where n is 5. In some cases, a linker comprises the aminoacid sequence (GGGGS)n (SEQ ID NO:205), where n is 1. In some cases, alinker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:206), wheren is 2. In some cases, a linker comprises the amino acid sequence(GGGGS)n (SEQ ID NO:207), where n is 3. In some cases, a linkercomprises the amino acid sequence (GGGGS)n (SEQ ID NO:208), where n is4. In some cases, a linker comprises the amino acid sequence (GGGGS)n(SEQ ID NO:209), where n is 5. In some cases, a linker comprises theamino acid sequence AAAGG (SEQ ID NO:73).

In some cases, a linker polypeptide, present in a first polypeptide of amultimeric polypeptide of the present disclosure, includes a cysteineresidue that can form a disulfide bond with a cysteine residue presentin a second polypeptide of a multimeric polypeptide of the presentdisclosure. In some cases, for example, a suitable linker comprises theamino acid sequence GCGASGGGGSGGGGS (SEQ ID NO:74).

Epitopes

An epitope (a peptide presenting one or more epitopes) present in amultimeric polypeptide of the present disclosure can have a length offrom about 4 amino acids to about 25 amino acids, e.g., the epitope canhave a length of from 4 amino acids (aa) to 10 aa, from 10 aa to 15 aa,from 15 aa to 20 aa, or from 20 aa to 25 aa. For example, an epitopepresent in a multimeric polypeptide of the present disclosure can have alength of 4 amino acids (aa), 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa. In some cases, an epitope present ina multimeric polypeptide of the present disclosure has a length of from5 amino acids to 10 amino acids, e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or10 aa.

An epitope present in a multimeric polypeptide of the present disclosureis specifically bound by a T-cell, i.e., the epitope is specificallybound by an epitope-specific T cell. An epitope-specific T cell binds anepitope having a reference amino acid sequence, but does notsubstantially bind an epitope that differs from the reference amino acidsequence. For example, an epitope-specific T cell binds an epitopehaving a reference amino acid sequence, and binds an epitope thatdiffers from the reference amino acid sequence, if at all, with anaffinity that is less than 10⁻⁶ M, less than 10⁻⁵ M, or less than 10⁻⁴M. An epitope-specific T cell can bind an epitope for which it isspecific with an affinity of at least 10⁻⁷ M, at least 10⁻⁸ M, at least10⁻⁹ M, or at least 10⁻¹⁰ M.

Suitable epitopes include, but are not limited to, epitopes present in acancer-associated antigen. Cancer-associated antigens include, but arenot limited to, α-folate receptor; carbonic anhydrase IX (CAIX); CD19;CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic antigen (CEA);epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40);folate binding protein (FBP); fetal acetylcholine receptor; gangliosideantigen GD2; Her2/neu; IL-13R-a2; kappa light chain; LeY; L1 celladhesion molecule; melanoma-associated antigen (MAGE); MAGE-A1;mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stemcell antigen (PSCA); prostate-specific membrane antigen (PSMA);tumor-associate glycoprotein-72 (TAG-72); and vascular endothelialgrowth factor receptor-2 (VEGF-R2). See, e.g., Vigneron et al. (2013)Cancer Immunity 13:15; and Vigneron (2015) BioMed Res. Int'l Article ID948501. In some cases, the epitope is a human papilloma virus E7 antigenepitope; see, e.g., Ramos et al. (2013) J. Immunother. 36:66.

In some cases, the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO:75).In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO:76). Insome cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO:77). Insome cases, the epitope is HPV16E7/11-19 (YMLDLQPET; SEQ ID NO:78). See,e.g., Ressing et al. ((1995) J. Immunol. 154:5934) for additionalsuitable HPV epitopes.

Immunomodulatory Polypeptides

Suitable immunomodulatory polypeptides include, but are not limited to,an IL-2 polypeptide, a 4-1BBL polypeptide, a B7-1 polypeptide; a B7-2polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasLpolypeptide, and a PD-L2 polypeptide.

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of a PD-L1 polypeptide depicted in FIG. 50A orFIG. 50B.

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of a CD80 polypeptide depicted in FIG. 51 .

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of an ICOS-L polypeptide depicted in FIG. 51 .

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of an OX40L polypeptide depicted in FIG. 53 .

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of a PD-L2 polypeptide depicted in FIG. 54 .

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of a CD86 polypeptide depicted in FIG. 55 .

In some cases, the immunomodulatory polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, at least 99%, or 100%, amino acid sequence identity withthe amino acid sequence of a FAS-L polypeptide depicted in FIG. 56 .

In some cases, the immunomodulatory polypeptide present in a synTacexhibits reduced binding affinity to a cognate co-immunomodulatorypolypeptide expressed on the surface of a T cell, compared to thebinding affinity of a wild-type immunomodulatory polypeptide for thesame cognate co-immunomodulatory polypeptide. In some cases, where asynTac comprises a reduced-affinity immunomodulatory polypeptide, thesynTac polypeptide exhibits reduced binding to a cognateco-immunomodulatory polypeptide expressed on the surface of a T cell.For example, in some cases, a synTac polypeptide that comprises areduced-affinity immunomodulatory polypeptid binds a cognateco-immunomodulatory polypeptide with a binding affinity that is at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50% less, at least 55% less,at least 60% less, at least 65% less, at least 70% less, at least 75%less, at least 80% less, at least 85% less, at least 90% less, at least95% less, or more than 95% less, than the binding affinity of a controlsynTac polypeptide comprising a wild-type immunomodulatory polypeptidefor the same cognate co-immunomodulatory polypeptide.

Determining Binding Affinity

Binding affinity between an immunomodulatory polypeptide and its cognateco-immunomodulatory polypeptide can be determined by bio-layerinterferometry (BLI) using purified immunomodulatory polypeptide andpurified cognate co-immunomodulatory polypeptide. Binding affinitybetween a synTac of the present disclosure and its cognateco-immunomodulatory polypeptide can also be determined by BLI usingpurified synTac and the cognate co-immunomodulatory polypeptide. BLImethods are well known to those skilled in the art. See, e.g., Lad etal. (2015) J. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014)J. Vis. Exp. 18:e51383. The specific and relative binding affinitiesdescribed in this disclosure between an immunomodulatory polypeptide andits cognate co-immunomodulatory polypeptide, or between a synTac and itscognate co-immunomodulatory polypeptide, can be determined using thefollowing procedures.

To determine binding affinity between a synTac of the present disclosureand its cognate co-immunomodulatory polypeptide, a BLI assay can becarried out using an Octet RED 96 (Pal FortéBio) instrument, or asimilar instrument, as follows. To determine binding affinity of aT-cell modulatory multimeric polypeptide (e.g., a synTac of the presentdisclosure; or a control T-cell modulatory multimeric polypeptide (wherea control T-cell modulatory multimeric polypeptide comprises a wild-typeimmunomodulatory polypeptide)), the T-cell modulatory multimericpolypeptide is immobilized onto an insoluble support (a “biosensor”).The immobilized T-cell modulatory multimeric polypeptide is the“target.” Immobilization can be effected by immobilizing a captureantibody onto the insoluble support, where the capture antibodyimmobilizes the T-cell modulatory multimeric polypeptide. For example,immobilization can be effected by immobilizing anti-Fc (e.g., anti-humanIgG Fc) antibodies onto the insoluble support, where the immobilizedanti-Fc antibodies bind to and immobilize the T-cell modulatorymultimeric polypeptide (where the T-cell modulatory multimericpolypeptide comprises an IgFc polypeptide). A co-immunomodulatorypolypeptide is applied, at several different concentrations, to theimmobilized T-cell modulatory multimeric polypeptide, and theinstrument's response recorded. Assays are conducted in a liquid mediumcomprising 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl,0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent.Binding of the co-immunomodulatory polypeptide to the immobilized T-cellmodulatory multimeric polypeptide is conducted at 30° C. As a positivecontrol for binding affinity, an anti-MHC Class I monoclonal antibodycan be used. For example, anti-HLA Class I monoclonal antibody W6/32(American Type Culture Collection No. HB-95; Parham et al. (1979) J.Immunol. 123:342), which has a K_(D) of 7 nM, can be used. A standardcurve can be generated using serial dilutions of the anti-MHC Class Imonoclonal antibody. The co-immunomodulatory polypeptide, or theanti-MHC Class I mAb, is the “analyte.” BLI analyzes the interferencepattern of white light reflected from two surfaces: i) from theimmobilized polypeptide (“target”); and ii) an internal reference layer.A change in the number of molecules (“analyte”; e.g.,co-immunomodulatory polypeptide; anti-HLA antibody) bound to thebiosensor tip causes a shift in the interference pattern; this shift ininterference pattern can be measured in real time. The two kinetic termsthat describe the affinity of the target/analyte interaction are theassociation constant (k_(a)) and dissociation constant (k_(d)). Theratio of these two terms (k_(d)/a) gives rise to the affinity constantK_(D).

As noted above, determining binding affinity between an immunomodulatorypolypeptide (e.g., IL-2 or an IL-2 variant) and its cognateco-immunomodulatory polypeptide (e.g., IL-2R) also can be determined byBLI. The assay is similar to that described above for the synTacmultimeric polypeptide. A BLI assay can be carried out using an OctetRED 96 (Pal FortéBio) instrument, or a similar instrument, as follows. Acomponent immunomodulatory polypeptide of a synTac of the presentdisclosure (e.g., a variant IL-2 polypeptide of the present disclosure);and a control immunomodulatory polypeptide (where a controlimmunomodulatory polypeptide comprises a wild-type immunomodulatorypolypeptide, e.g. wild-type IL-2)) are immobilized onto an insolublesupport (a “biosensor”). The immunomodulatory polypeptide is the“target.” Immobilization can be effected by immobilizing a captureantibody onto the insoluble support, where the capture antibodyimmobilizes the immunomodulatory polypeptide. For example, if the targetis fused to an immuno-affinity tag (e.g. FLAG, human IgG Fc)immobilization can be effected by immobilizing with the appropriateantibody to the immuno-affinity tag (e.g. anti-human IgG Fc) onto theinsoluble support, where the immobilized antibodies bind to andimmobilize the immunomodulatory polypeptide (where the immunomodulatorypolypeptide comprises an IgFc polypeptide). A co-immunomodulatorypolypeptide (or polypeptides) is applied, at several differentconcentrations, to the immobilized immunomodulatory polypeptide, and theinstrument's response recorded. Alternatively, a co-immunomodulatorypolypeptide (or polypeptides) is immobilized to the biosensor (e.g., forthe IL-2 receptor heterotrimer, as a monomeric subunit, heterodimericsubcomplex, or the complete heterotrimer) and the immunomodulatorypolypeptide is applied, at several different concentrations, to theimmoobilized coimmunomodulatory polypeptide(s), and the instrument'sresponse is recorded. Assays are conducted in a liquid medium comprising25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1%bovine serum albumin, and 0.02% Tween 20 nonionic detergent. Binding ofthe co-immunomodulatory polypeptide to the immobilized immunomodulatorypolypeptide is conducted at 30° C. As a positive control for bindingaffinity, an anti-MHC Class I monoclonal antibody can be used. Forexample, anti-HLA Class I monoclonal antibody W6/32 (American TypeCulture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342),which has a K_(D) of 7 nM, can be used. A standard curve can begenerated using serial dilutions of the anti-MHC Class I monoclonalantibody. The co-immunomodulatory polypeptide, or the anti-MHC Class ImAb, is the “analyte.” BLI analyzes the interference pattern of whitelight reflected from two surfaces: i) from the immobilized polypeptide(“target”); and ii) an internal reference layer. A change in the numberof molecules (“analyte”; e.g., co-immunomodulatory polypeptide; anti-HLAantibody) bound to the biosensor tip causes a shift in the interferencepattern; this shift in interference pattern can be measured in realtime. The two kinetic terms that describe the affinity of thetarget/analyte interaction are the association constant (k_(a)) anddissociation constant (k_(d)). The ratio of these two terms (k_(d)/a)gives rise to the affinity constant K_(D). Determining the bindingaffinity of both a wild-type immunomodulatory polypeptide (e.g., IL-2)for its receptor (e.g., IL-2R) and a variant immunomodulatorypolypeptide (e.g., an IL-2 variant as disclosed herein) for its cognateco-immunomodulatory polypeptide (e.g., its receptor) (e.g., IL-2R) thusallows one to determine the relative binding affinity of the variantco-immunomodulatory polypeptide, as compared to the wild-typeco-immunomodulatory polypeptide, for the cognate co-immunomodulatorypolypeptide. That is, one can determine whether the binding affinity ofa variant immunomodulatory polypetpide for its receptor (its cognateco-immunomodulatory polypeptide) is reduced as compared to the bindingaffinity of the wild-type immunomodulatory polypeptide for the samecognate co-immunomodulatory polypeptide, and, if so, what is thepercentage reduction from the binding affinity of the wild-typeco-immunomodulatory polypeptide.

The BLI assay is carried out in a multi-well plate. To run the assay,the plate layout is defined, the assay steps are defined, and biosensorsare assigned in Octet Data Acquisition software. The biosensor assemblyis hydrated. The hydrated biosensor assembly and the assay plate areequilibrated for 10 minutes on the Octet instrument. Once the data areacquired, the acquired data are loaded into the Octet Data Analysissoftware. The data are processed in the Processing window by specifyingmethod for reference subtraction, y-axis alignment, inter-stepcorrection, and Savitzky-Golay filtering. Data are analyzed in theAnalysis window by specifying steps to analyze (Association andDissociation), selecting curve fit model (1:1), fitting method (global),and window of interest (in seconds). The quality of fit is evaluated.K_(D) values for each data trace (analyte concentration) can be averagedif within a 3-fold range. K_(D) error values should be within one orderof magnitude of the affinity constant values; R² values should be above0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

In some cases, the ratio of: i) the binding affinity of a control T-cellmodulatory multimeric polypeptide (where the control comprises awild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to acognate co-immunomodulatory polypeptide (e.g., IL-2R) to ii) the bindingaffinity of a T-cell modulatory multimeric polypeptide of the presentdisclosure comprising a variant of the wild-type immunomodulatorypolypeptide (e.g., variant IL-2) to the cognate co-immunomodulatorypolypeptide (e.g., IL-2R), when measured by BLI (as described above), isat least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, atleast 500:1, at least 10²:1, at least 5×10²:1, at least 10³:1, at least5×10³:1, at least 104:1, at least 105:1, or at least 10⁶:1. In somecases, the ratio of: i) the binding affinity of a control T-cellmodulatory multimeric polypeptide (where the control comprises awild-type immunomodulatory polypeptide) to a cognate co-immunomodulatorypolypeptide to ii) the binding affinity of a T-cell modulatorymultimeric polypeptide of the present disclosure comprising a variant ofthe wild-type immunomodulatory polypeptide to the cognateco-immunomodulatory polypeptide, when measured by BLI, is in a range offrom 1.5:1 to 106:1, e.g., from 1.5:1 to 10:1, from 10:1 to 50:1, from50:1 to 10²:1, from 10²:1 to 10¹:1, from 10³:1 to 104:1, from 104:1 to105:1, or from 105:1 to 10⁶:1.

In some cases, the ratio of: i) the binding affinity of a controlimmunomodulatory polypeptide (where the control comprises a wild-typeimmunomodulatory polypeptide, e.g., wild-type IL-2) to a cognateco-immunomodulatory polypeptide (e.g., IL-2R) to ii) the bindingaffinity of a immunomodulatory polypeptide of the present disclosurecomprising a variant of the wild-type immunomodulatory polypeptide(e.g., variant IL-2) to the cognate co-immunomodulatory polypeptide(e.g., IL-2R), when measured by BLI (as described above), is at least1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, atleast 20:1, at least 25:1, at least 50:1, at least 100:1, at least500:1, at least 10²:1, at least 5×10²:1, at least 10³:1, at least5×10³:1, at least 10⁴:1, at least 10⁵:1, or at least 10⁶:1. In somecases, the ratio of: i) the binding affinity of a controlimmunomodulatory polypeptide (where the control comprises a wild-typeimmunomodulatory polypeptide) to a cognate co-immunomodulatorypolypeptide to ii) the binding affinity of a immunomodulatorypolypeptide of the present disclosure comprising a variant of thewild-type immunomodulatory polypeptide to the cognateco-immunomodulatory polypeptide, when measured by BLI, is in a range offrom 1.5:1 to 106:1, e.g., from 1.5:1 to 10:1, from 10:1 to 50:1, from50:1 to 10²:1, from 10²:1 to 10³:1, from 10³:1 to 10⁴:1, from 10⁴:1 to10⁵:1, or from 10⁵:1 to 10⁶:1.

IL-2/synTac

In some cases, a multimeric polypeptide comprises a wild-type(naturally-occurring) IL-2 as the modulatory domain. In some cases, amultimeric polypeptide comprises a variant IL-2 polypeptide as themodulatory domain.

A T-cell modulatory multimeric polypeptide that comprises an IL-2polypeptide as the modulatory (“MOD”) domain is also referred to as an“IL-2/synTac,” “an IL-2/synTac polypeptide” or an “IL-2/multimericpolypeptide.”

In some cases, an IL-2/synTac polypeptide comprises a wild-type IL-2polypeptide. In some cases, a synTac polypeptide comprises a single copyof a wild-type IL-2 polypeptide. In some cases, a synTac polypeptidecomprises two copies of a wild-type IL-2 polypeptide. In some cases, asynTac polypeptide comprises three copies of a wild-type IL-2polypeptide. In some cases, the wild-type IL-2 polypeptide comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2A. A wild-type amino acid sequence of a human IL2polypeptide can be as follows: APTSSSTKKT QLQLEHLLLD LQMILNGINNYKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNINVIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1).

In some cases, a synTac polypeptide comprises a variant IL-2polypeptide. A variant IL-2 polypeptide present in a multimericpolypeptide exhibits reduced binding affinity to an IL2R, compared tothe binding affinity of wild-type IL-2 to the IL2R. A multimericpolypeptide that comprises a variant IL-2 polypeptide also exhibitsreduced binding affinity for an IL2R, compared to a control multimericpolypeptide comprising a wild-type IL-2 for IL2R (e.g., an IL2Rcomprising alpha, beta, and gamma polypeptides comprising the amino acidsequences (mature form) depicted in FIG. 3A-3C).

In some cases, an IL-2/synTac polypeptide exhibits reduced bindingaffinity to IL2R, compared to the binding affinity of an IL2 polypeptidecomprising the amino acid sequence depicted in FIG. 2A for IL2R. Forexample, in some cases, an IL-2/synTac polypeptide binds IL2R with abinding affinity that is less than the binding affinity of a controlsynTac polypeptide comprising an IL2 polypeptide comprising the aminoacid sequence depicted in FIG. 2A for an IL2R comprising alpha, beta,and gamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C. For example, in some cases, an IL-2/synTacpolypeptide binds IL2R with a binding affinity that is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50% less, at least 55% less, at least60% less, at least 65% less, at least 70% less, at least 75% less, atleast 80% less, at least 85% less, at least 90% less, at least 95% less,or more than 95% less, than the binding affinity of a control synTacpolypeptide comprising an IL-2 polypeptide comprising the amino acidsequence depicted in FIG. 2A for IL2R (e.g., an IL2R comprising alpha,beta, and gamma polypeptides comprising the amino acid sequences (matureform) depicted in FIG. 3A-3C).

In some cases, an IL-2/synTac polypeptide has a binding affinity forIL2R that is from 100 nm to about 100 μM. In some cases, an IL-2/synTacpolypeptide has a binding affinity for IL2R that is from about 100 nM to500 nM. For example, in some cases, an IL-2/synTac polypeptide has abinding affinity for IL2R (e.g., an IL2R comprising alpha, beta, andgamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C) that is from about 100 nM to about 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 450 nM, or fromabout 450 nM to about 500 nM. In some cases, an IL-2/synTac polypeptidehas a binding affinity for IL2R (e.g., an IL2R comprising alpha, beta,and gamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C) that is from about 500 nM to 1 M. For example,in some cases, an IL-2/synTac polypeptide has a binding affinity forIL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptidescomprising the amino acid sequences (mature form) depicted in FIG.3A-3C) that is from about 500 nM to about 600 nM, from about 600 nM toabout 700 nM, from about 700 nM to about 800 nM, from about 800 nM toabout 900 nM, or from about 900 nM to about 1 μM. In some cases, anIL-2/synTac polypeptide has a binding affinity for IL2R (e.g., an IL2Rcomprising alpha, beta, and gamma polypeptides comprising the amino acidsequences (mature form) depicted in FIG. 3A-3C) that is from about 1 μMto 10 μM. For example, in some cases, an IL-2/synTac polypeptide has abinding affinity for IL2R (e.g., an IL2R comprising alpha, beta, andgamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C) that is from about 1 μM to 2 μM, from about 2 μMto about 3 μM, from about 3 μM to about 4 μM, from about 4 μM to about 5μM, from about 5 μM to about 6 μM, from about 6 μM to about 7 μM, fromabout 7 μM to about 8 μM, from about 8 μM to about 9 μM, or from about 9μM to about 10 μM. In some cases, an IL-2/synTac polypeptide has abinding affinity for IL2R (e.g., an IL2R comprising alpha, beta, andgamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C) that is from about 10 μM to 100 μM. For example,in some cases, an IL-2/synTac polypeptide has a binding affinity forIL2R (e.g., an IL2R comprising alpha, beta, and gamma polypeptidescomprising the amino acid sequences (mature form) depicted in FIG.3A-3C) that is from about 10 μM to about 20 μM, from about 20 μM toabout 30 μM, from about 30 μM to about 40 μM, from about 40 μM to about50 μM, from about 50 μM to about 60 μM, from about 60 μM to about 70 μM,from about 70 μM to about 80 μM, from about 80 μM to about 90 μM, orfrom about 90 μM to about 100 μM.

A variant IL2 polypeptide present in an IL-2/synTac polypeptide can havea single amino acid substitution relative to a wild-type IL2 polypeptide(e.g., a IL2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL2polypeptide present in an IL-2/synTac polypeptide has from 2 to 10 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 2 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 3 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 4 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 5 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 6 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 7 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 8 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 9 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL2 polypeptidepresent in a synTac polypeptide of the present disclosure has 10 aminoacid substitutions relative to a wild-type IL2 polypeptide (e.g., a IL2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1).

In some cases, a multimeric polypeptide of the present disclosurecomprises a first polypeptide and a second polypeptide, where the firstpolypeptide comprises, in order from amino terminus (N-terminus) tocarboxyl terminus (C-terminus): a) an epitope (e.g., a T-cell epitope);b) a first major histocompatibility complex (MHC) polypeptide and c) animmunomodulatory polypeptide (e.g., a variant IL2 polypeptide of thepresent disclosure); and where the second polypeptide comprises, inorder from N-terminus to C-terminus: a) a second MHC polypeptide; and b)an immunoglobulin (Ig) Fc polypeptide. In other cases, a multimericpolypeptide of the present disclosure comprises a first polypeptide anda second polypeptide, where the first polypeptide comprises, in orderfrom N-terminus to C-terminus: a) an epitope (e.g., a T-cell epitope);and b) a first MHC polypeptide; and where the second polypeptidecomprises, in order from N-terminus to C-terminus: a) animmunomodulatory polypeptide (e.g., a variant IL2 polypeptide of thepresent disclosure); b) a second MHC polypeptide; and c) an Ig Fcpolypeptide. In some instances, the first and the second MHCpolypeptides are Class I MHC polypeptides; e.g., in some cases, thefirst MHC polypeptide is an MHC Class I 02-microglobulin (B2M or β2M)polypeptide, and the second MHC polypeptide is an MHC Class I heavychain (H chain); or the first MHC polypeptide is an MHC Class I H chain,and the second MHC polypeptide is an MHC Class I β2M polypeptide). Inother cases, the first and the second MHC polypeptides are Class II MHCpolypeptides; e.g., in some cases, the first MHC polypeptide is an MHCClass II α-chain polypeptide, and the second MHC polypeptide is an MHCClass II β-chain polypeptide. In other cases, the first polypeptide isan MHC Class II β-chain polypeptide, and the second MHC polypeptide isan MHC Class II α-chain polypeptide. In some cases, the multimericpolypeptide includes two or more immunomodulatory polypeptides, where atleast one of the immunomodulatory polypeptides is a variant IL2immunomodulatory polypeptide of the present disclosure. Where amultimeric polypeptide of the present disclosure includes two or moreimmunomodulatory polypeptides, in some cases, the two or moreimmunomodulatory polypeptides are present in the same polypeptide chain,and may be in tandem. Where a multimeric polypeptide of the presentdisclosure includes two or more immunomodulatory polypeptides, in somecases, the two or more immunomodulatory polypeptides are present inseparate polypeptides. In some cases, a multimeric polypeptide of thepresent disclosure is a heterodimer. In some cases, a multimericpolypeptide of the present disclosure is a trimeric polypeptide.

In some cases, a multimeric polypeptide comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; and ii) a first MHC polypeptide; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus: i) a second MHCpolypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatorydomain (e.g., a variant IL2 polypeptide of the present disclosure). Insome cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) asecond polypeptide comprising, in order from N-terminus to C-terminus:i) a second MHC polypeptide; and ii) an immunomodulatory domain (e.g., avariant IL2 polypeptide of the present disclosure). In some cases, amultimeric polypeptide of the present disclosure comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; and ii) a first MHC polypeptide; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus: i) animmunomodulatory domain (e.g., a variant IL2 polypeptide of the presentdisclosure); and ii) a second MHC polypeptide. In some cases, amultimeric polypeptide of the present disclosure comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; ii) a first MHC polypeptide; and iii) an immunomodulatorydomain (e.g., a variant IL2 polypeptide of the present disclosure); andb) a second polypeptide comprising, in order from N-terminus toC-terminus: i) a second MHC polypeptide. In some cases, where amultimeric polypeptide of the present disclosure comprises a non-Igscaffold, the non-Ig scaffold is an XTEN peptide, a transferrinpolypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, asilk-like polypeptide, or a silk-elastin-like polypeptide.

In some cases, a multimeric polypeptide of the present disclosure ismonovalent. In some cases, a multimeric polypeptide of the presentdisclosure is multivalent. In some cases, a multivalent multimericpolypeptide of the present disclosure comprises an immunoglobulin Fcpolypeptide on one of the first or the second polypeptide. For example,depending on the Fc polypeptide present in a multimeric polypeptide ofthe present disclosure, the multimeric polypeptide can be a homodimer,where two molecules of the multimeric polypeptide are present in thehomodimer, where the two molecules of the multimeric polypeptide can bedisulfide linked to one another, e.g., via the Fc polypeptide present inthe two molecules. As another example, a multimeric polypeptide of thepresent disclosure can comprise three, four, or five molecules of themultimeric polypeptide, where the molecules of the multimericpolypeptide can be disulfide linked to one another, e.g., via the Fcpolypeptide present in the molecules.

In some cases, a multimeric polypeptide comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; ii) a β2M polypeptide; and iii) a variant IL2 polypeptide ofthe present disclosure; and b) a second polypeptide comprising, in orderfrom N-terminus to C-terminus: i) a Class I MHC heavy chain; and ii) anFc polypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus: i) an epitope; and ii) a β2M polypeptide; andb) a second polypeptide comprising, in order from N-terminus toC-terminus: i) a variant IL2 polypeptide of the present disclosure; ii)a Class I MHC heavy chain; and iii) an Fc polypeptide. In some cases, amultimeric polypeptide of the present disclosure comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; ii) a β2M polypeptide; iii) a first variant IL2 polypeptide ofthe present disclosure; iv) a second variant IL2 polypeptide of thepresent disclosure; and v) a third variant IL2 polypeptide of thepresent disclosure; and b) a second polypeptide comprising, in orderfrom N-terminus to C-terminus: i) a Class I MHC heavy chain; and ii) anFc polypeptide. In some cases, the first, second, and third variant IL2polypeptides have the same amino acid sequence. In some cases, thefirst, second, and third variant IL2 polypeptides differ from oneanother in amino acid sequence. In some cases, a multimeric polypeptideof the present disclosure comprises: a) a first polypeptide comprising,in order from N-terminus to C-terminus: i) an epitope; and ii) a β2Mpolypeptide; and b) a second polypeptide comprising, in order fromN-terminus to C-terminus: i) a first variant IL2 polypeptide of thepresent disclosure; ii) a second variant IL2 polypeptide of the presentdisclosure; and iii) a third variant IL2 polypeptide of the presentdisclosure; iv) a Class I MHC heavy chain; and v) an Fc polypeptide. Insome cases, the first, second, and third variant IL2 polypeptides havethe same amino acid sequence. In some cases, the first, second, andthird variant IL2 polypeptides differ from one another in amino acidsequence.

F42 Substitution

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2B, where amino acid 42 isan amino acid other than a phenylalanine, e.g., where amino acid 42 isGly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2B, where amino acid 42 is Ala, Gly, Val, Leu, or Ile.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2B,where amino acid 42 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2B, where amino acid 42 is Gly. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2B, where aminoacid 42 is Val. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2B, where amino acid 42 is Leu. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2B, where amino acid 42 isIle. In some cases, a single copy of the variant IL-2 polypeptide ispresent in a multimeric polypeptide of the present disclosure. In somecases, a multimeric polypeptide of the present disclosure comprises twocopies of the variant IL-2 polypeptide, e.g., where the two copies arein tandem with no linker between the two copies, or are in tandem andseparated by a linker peptide. In some cases, a multimeric polypeptideof the present disclosure comprises three copies of the variant IL-2polypeptide, e.g., where the three copies are in tandem with no linkerbetween the three copies, or are in tandem and separated by a linkerpeptide. In some cases, where an IL-2/synTac of the present disclosurecomprises HLA Class I heavy chain and β2M, the IL-2 polypeptide(s)is/are on the polypeptide chain comprising the HLA Class I heavy chain.In some cases, where an IL-2/synTac of the present disclosure comprisesHLA Class I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the β2M polypeptide. In some cases, thevariant IL-2 polypeptide, or the synTac comprising same, has a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about M, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, the variant IL-2 polypeptide present in a multimeric polypeptideof the present disclosure has a length of 133 amino acids.

Y45 Substitution

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2F,where amino acid 45 is an amino acid other than a tyrosine, e.g., whereamino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2F, where amino acid 45 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2F, where amino acid 45 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2F, where aminoacid 45 is Gly. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2F, where amino acid 45 is Val. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2F, where amino acid 45 isLeu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2F,where amino acid 45 is Ile. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or the synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about M, from about 5 μM to about 10 μM, from about 10μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM toabout 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about75 μM, or from about 75 μM to about 100 μM. In some cases, the variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure has a length of 133 amino acids.

Q126 Substitution

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2G,where amino acid 126 is an amino acid other than a glutamine, e.g.,where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2G, where amino acid 126 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2G,where amino acid 126 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2G, where amino acid 126 is Val. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Leu. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2G, where amino acid 126 is Ile. In some cases, a single copy ofthe variant IL-2 polypeptide is present in a multimeric polypeptide ofthe present disclosure. In some cases, a multimeric polypeptide of thepresent disclosure comprises two copies of the variant IL-2 polypeptide,e.g., where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about M, from about 5 μM to about 10 μM, from about 10μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM toabout 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about75 μM, or from about 75 μM to about 100 μM. In some cases, the variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure has a length of 133 amino acids.

F42 and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2H,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; and where amino acid 16is an amino acid other than a histidine, e.g., where amino acid 16 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, Asp, or Glu. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2H, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2H, where aminoacid 42 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2H, where amino acid 42 isAla and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2H, where amino acid 42 is Val and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2H,where amino acid 42 is Leu, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2H, where aminoacid 42 is Ile and amino acid 16 is Ala. In some cases, a single copy ofthe variant IL-2 polypeptide is present in a multimeric polypeptide ofthe present disclosure. In some cases, a multimeric polypeptide of thepresent disclosure comprises two copies of the variant IL-2 polypeptide,e.g., where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, a multimeric polypeptideof the present disclosure comprises 2 copies of the IL-2 variantcomprising F42A and H16A substitutions, where the multimeric polypeptidecomprises HLA Class I heavy chain and β2M polypeptides, and where the 2copies of IL-2 (F42A, H16A) are on the polypeptide chain comprising theHLA Class I heavy chain. In some cases, the variant IL-2 polypeptide, ora synTac comprising same, has a binding affinity for IL2R that is fromabout 100 nM to 150 nM, from about 150 nM to about 200 nM, from about200 nM to about 250 nM, from about 250 nM to about 300 nM, from about300 nM to about 350 nM, from about 350 nM to about 400 nM, from about400 nM to about 500 nM, from about 500 nM to about 600 nM, from about600 nM to about 700 nM, from about 700 nM to about 800 nM, from about800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μMto about 5 μM, from about 5 M to about 10 μM, from about 10 μM to about15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 M, or fromabout 75 μM to about 100 μM. In some cases, the variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure has alength of 133 amino acids. In some cases, the variant IL-2 polypeptidecomprises the amino acid sequence depicted in FIG. 34B (comprising H16Aand F42A substitutions).

F42 and D20 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21 ,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; and where amino acid 20is an amino acid other than an aspartic acid, e.g., where amino acid 20is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21 , where amino acid 42 is Ala, Gly, Val, Leu, or Ile;and where amino acid 20 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 21 , where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; and where amino acid 20 is Asn,Gln, Lys, Arg, or His. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21 , where amino acid 42 is Ala and amino acid 20 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21 ,where amino acid 42 is Ala and amino acid 20 is Gly. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 21 , where aminoacid 42 is Val and amino acid 20 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 21 , where amino acid 42 isLeu, and amino acid 20 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21 , where amino acid 42 is Ile and amino acid 20 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21 ,where amino acid 42 is Ala and amino acid 20 is Asn. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 21 , where aminoacid 42 is Ala and amino acid 20 is Gln. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 21 , where amino acid 42 isAla and amino acid 20 is Lys. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21 , where amino acid 42 is Ala and amino acid 20 isArg. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21 ,where amino acid 42 is Ala and amino acid 20 is His. In some cases, asingle copy of the variant IL-2 polypeptide is present in a multimericpolypeptide of the present disclosure. In some cases, a multimericpolypeptide of the present disclosure comprises two copies of thevariant IL-2 polypeptide, e.g., where the two copies are in tandem withno linker between the two copies, or are in tandem and separated by alinker peptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, where an IL-2/synTac of the present disclosure comprises HLAClass I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the HLA Class I heavy chain. In some cases,where an IL-2/synTac of the present disclosure comprises HLA Class Iheavy chain and β2M, the IL-2 polypeptide(s) is/are on the polypeptidechain comprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, and E15 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 15 is an amino acidother than a glutamic acid, e.g., where amino acid 15 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg,His, or Asp. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2J, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu,or Ile; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2J, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln,Lys, Arg, or His; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 isGly. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 isGly. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 isGly. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21 ,where amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21 ,where amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 isAla. In some cases, a single copy of the variant IL-2 polypeptide ispresent in a multimeric polypeptide of the present disclosure. In somecases, a multimeric polypeptide of the present disclosure comprises twocopies of the variant IL-2 polypeptide, e.g., where the two copies arein tandem with no linker between the two copies, or are in tandem andseparated by a linker peptide. In some cases, where an IL-2/synTac ofthe present disclosure comprises HLA Class I heavy chain and β2M, theIL-2 polypeptide(s) is/are on the polypeptide chain comprising the HLAClass I heavy chain. In some cases, where an IL-2/synTac of the presentdisclosure comprises HLA Class I heavy chain and β2M, the IL-2polypeptide(s) is/are on the polypeptide chain comprising the β2Mpolypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, the variant IL-2 polypeptide, or a synTac comprising same, has abinding affinity for IL2R that is from about 100 nM to 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 500 nM, fromabout 500 nM to about 600 nM, from about 600 nM to about 700 nM, fromabout 700 nM to about 800 nM, from about 800 nM to about 900 nM, fromabout 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μMto about 10 M, from about 10 μM to about 15 μM, from about 15 μM toabout 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100μM. In some cases, the variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure has a length of 133 amino acids.

F42, D20, and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2K,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 16 is an amino acidother than a histidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, orGlu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2K,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2K, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 20 is Asn, Gln, Lys, Arg, or His; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Gly,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Leu, amino acid 20 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Asn,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Lys,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is His,and amino acid 16 is Ala. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 126 is an amino acidother than a glutamine, e.g., where amino acid 126 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His,Asp, or Glu. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2L, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 20 is Ala, Gly, Val, Leu, or Ile; and where amino acid 126 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2L, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;where amino acid 20 is Asn, Gln, Lys, Arg, or His; and where amino acid126 is Ala, Gly, Val, Leu, or Ile. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2L, where amino acid 42 isAla, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ile, amino acid 20 is Ala, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In somecases, a single copy of the variant IL-2 polypeptide is present in amultimeric polypeptide of the present disclosure. In some cases, amultimeric polypeptide of the present disclosure comprises two copies ofthe variant IL-2 polypeptide, e.g., where the two copies are in tandemwith no linker between the two copies, or are in tandem and separated bya linker peptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, where an IL-2/synTac of the present disclosure comprises HLAClass I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the HLA Class I heavy chain. In some cases,where an IL-2/synTac of the present disclosure comprises HLA Class Iheavy chain and β2M, the IL-2 polypeptide(s) is/are on the polypeptidechain comprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, and Y45 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2M,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 45 is an amino acidother than a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, orGlu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2M,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 45 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2M, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 20 is Asn, Gln, Lys, Arg, or His; and where amino acid 45 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Gly,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Leu, amino acid 20 is Ala,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Asn,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Lys,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is His,and amino acid 45 is Ala. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F4, D20, Y45, and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; where amino acid 45 is an amino acid otherthan a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu;and where amino acid 16 is an amino acid other than a histidine, e.g.,where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu,or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; and whereamino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg,or His; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; and whereamino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Val, amino acid 20 is Ala,amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isLeu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 isVal. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Ala, amino acid 20 is Asn,amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Ala, amino acid 20 is Arg,amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is His, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a single copy of the variant IL-2 polypeptide ispresent in a multimeric polypeptide of the present disclosure. In somecases, a multimeric polypeptide of the present disclosure comprises twocopies of the variant IL-2 polypeptide, e.g., where the two copies arein tandem with no linker between the two copies, or are in tandem andseparated by a linker peptide. In some cases, a multimeric polypeptideof the present disclosure comprises three copies of the variant IL-2polypeptide, e.g., where the three copies are in tandem with no linkerbetween the three copies, or are in tandem and separated by a linkerpeptide. In some cases, where an IL-2/synTac of the present disclosurecomprises HLA Class I heavy chain and β2M, the IL-2 polypeptide(s)is/are on the polypeptide chain comprising the HLA Class I heavy chain.In some cases, where an IL-2/synTac of the present disclosure comprisesHLA Class I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the β2M polypeptide. In some cases, thevariant IL-2 polypeptide, or a synTac comprising same, has a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, the variant IL-2 polypeptide present in a multimeric polypeptideof the present disclosure has a length of 133 amino acids.

F42, D20, Y45, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2O,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; where amino acid 45 is an amino acid otherthan a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu;and where amino acid 126 is an amino acid other than a glutamine, e.g.,where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly,Val, Leu, or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; andwhere amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln,Lys, Arg, or His; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; andwhere amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2O, where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45is Gly, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2O, where amino acid 42 isVal, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 126 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2O,where amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly,and amino acid 126 is Val. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2O, where amino acid 42 is Ile, amino acid 20 is Ala,amino acid 45 is Ala, and amino acid 126 is Gly. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2O, where amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45is Ala, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2O, where amino acid 42 isAla, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 126 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2O,where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala,and amino acid 126 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2O, where amino acid 42 is Ala, amino acid 20 is His,amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, a singlecopy of the variant IL-2 polypeptide is present in a multimericpolypeptide of the present disclosure. In some cases, a multimericpolypeptide of the present disclosure comprises two copies of thevariant IL-2 polypeptide, e.g., where the two copies are in tandem withno linker between the two copies, or are in tandem and separated by alinker peptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, where an IL-2/synTac of the present disclosure comprises HLAClass I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the HLA Class I heavy chain. In some cases,where an IL-2/synTac of the present disclosure comprises HLA Class Iheavy chain and β2M, the IL-2 polypeptide(s) is/are on the polypeptidechain comprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, Y45, H16, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; where amino acid 45 is an amino acid otherthan a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu;where amino acid 126 is an amino acid other than a glutamine, e.g.,where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; and where aminoacid 16 is an amino acid other than a histidine, e.g., where amino acid16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu,or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; where aminoacid 126 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;where amino acid 20 is Asn, Gln, Lys, Arg, or His; where amino acid 45is Ala, Gly, Val, Leu, or Ile; where amino acid 126 is Ala, Gly, Val,Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. Insome cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala,amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isVal, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Leu, amino acid 20 is Ala,amino acid 45 is Gly, amino acid 126 is Val, and amino acid 16 is Ala.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala,amino acid 126 is Gly, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isAla, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Ala, amino acid 20 is Lys,amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala,amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a single copy ofthe variant IL-2 polypeptide is present in a multimeric polypeptide ofthe present disclosure. In some cases, a multimeric polypeptide of thepresent disclosure comprises two copies of the variant IL-2 polypeptide,e.g., where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, Q126, and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2Q,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 126 isan amino acid other than a glutamine, e.g., where amino acid 126 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys,Arg, His, Asp, or Glu; and where amino acid 16 is an amino acid otherthan a histidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2Q,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 126is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2Q, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 126 is Asn, Gln, Lys, Arg, or His; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Gly,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Val, amino acid 126 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Leu, amino acid 126 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ile, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Asn,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Lys,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Arg,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is His,and amino acid 16 is Ala. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising the variant IL-2 polypeptide, has abinding affinity for IL2R that is from about 100 nM to 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 500 nM, fromabout 500 nM to about 600 nM, from about 600 nM to about 700 nM, fromabout 700 nM to about 800 nM, from about 800 nM to about 900 nM, fromabout 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μMto about 10 M, from about 10 μM to about 15 μM, from about 15 μM toabout 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100μM. In some cases, the variant IL-2 polypeptide has a length of 133amino acids. 4-1BBL

In some cases, a synTac suitable for use in a method of the presentdisclosure comprises a 4-1BBL polypeptide as the immunomodulatorydomain(s). Suitable 4-1BBL immunomodulatory domains include a wild-type4-1BBL immunomodulatory domain, and a variant 4-1BBL immunomodulatorydomain.

A wild-type human 4-1BBL amino acid sequence is provided in FIG. 36A.The tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBLcomprises amino acids 81-254, amino acids 80-254, or amino acids 80-246of the amino acid sequence depicted in FIG. 36A. Thus, a wild-type aminoacid sequence of the THD of human 4-1BBL can be, e.g., one of SEQ IDNOs:213-215, as follows:

(SEQ ID NO: 213) PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSLTGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGSVSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQGRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE.(SEQ ID NO: 214) D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSLTGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGSVSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQGRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE.(SEQ ID NO: 215) D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSLTGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGSVSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQGRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA

Wild-type 4-1BBL binds to 4-1BB (CD137). An amino acid sequences of4-1BB is provided in FIG. 37 . A variant 4-1BBL polypeptide of thepresent disclosure binds to 4-1BB with reduced affinity compared tobinding of wild-type 4-1BBL to 4-1BB.

Variant 4-1BBL polypeptides include those having an amino acid sequencethat has at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% amino acid sequence identity to a corresponding wild-type4-1BBL polypeptide, and include variant 4-1BBL polypeptides that differby 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, ormore than 15 amino acids, relative to a corresponding wild-type 4-1BBLpolypeptide. In some cases, a variant 4-1BBL polypeptide differs inamino acid sequence from a wild-type 4-1BBL polypeptide by only a singleamino acid. In some cases, a variant 4-1BBL polypeptide differs in aminoacid sequence from a wild-type 4-1BBL polypeptide by no more than 2amino acids. In some cases, a variant 4-1BBL polypeptide differs inamino acid sequence from a wild-type 4-1BBL polypeptide by no more than3 amino acids. In some cases, a variant 4-1BBL polypeptide differs inamino acid sequence from a wild-type 4-1BBL polypeptide by no more than4 amino acids. In some cases, a variant 4-1BBL polypeptide differs inamino acid sequence from a wild-type 4-1BBL polypeptide by no more than5 amino acids.

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure exhibits reduced binding affinityto 4-1BB, compared to the binding affinity of a 4-1BBL polypeptidecomprising the amino acid sequence depicted in FIG. 36A for 4-1BB. Forexample, in some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure binds 4-1BB with abinding affinity that is less than the binding affinity of a 4-1BBLpolypeptide comprising the amino acid sequence depicted in FIG. 36A fora 4-1BB polypeptide comprising the amino acid sequence depicted in FIG.37 . For example, in some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure binds 4-1BB with abinding affinity that is at least 10% less, at least 15% less, at least20% less, at least 25% less, at least 30% less, at least 35% less, atleast 40% less, at least 45% less, at least 50% less, at least 55% less,at least 60% less, at least 65% less, at least 70% less, at least 75%less, at least 80% less, at least 85% less, at least 90% less, at least95% less, or more than 95% less, than the binding affinity of a 4-1BBLpolypeptide comprising the amino acid sequence depicted in FIG. 36A for4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequencedepicted in FIG. 37 ).

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure exhibits reduced binding affinityto 4-1BB, compared to the binding affinity of a 4-1BBL polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:213 for 4-1BB.For example, in some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure binds 4-1BB with abinding affinity that is less than the binding affinity of a 4-1BBLpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:213for a 4-1BB polypeptide comprising the amino acid sequence depicted inone of FIG. 37A-37C. For example, in some cases, the variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure binds 4-1BB with a binding affinity that is at least 10%less, at least 15% less, at least 20% less, at least 25% less, at least30% less, at least 35% less, at least 40% less, at least 45% less, atleast 50% less, at least 55% less, at least 60% less, at least 65% less,at least 70% less, at least 75% less, at least 80% less, at least 85%less, at least 90% less, at least 95% less, or more than 95% less, thanthe binding affinity of a 4-1BBL polypeptide comprising the amino acidsequence depicted in SEQ ID NO:213 for 4-1BB (e.g., a 4-1BB polypeptidecomprising the amino acid sequence depicted in FIG. 37 ).

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure has a binding affinity to 4-1BBthat is from 100 nM to 100 μM. As another example, in some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure has a binding affinity for 4-1BB (e.g., a 4-1BBpolypeptide comprising the amino acid sequence depicted in FIG. 37 )that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM,from about 200 nM to about 250 nM, from about 250 nM to about 300 nM,from about 300 nM to about 350 nM, from about 350 nM to about 400 nM,from about 400 nM to about 500 nM, from about 500 nM to about 600 nM,from about 600 nM to about 700 nM, from about 700 nM to about 800 nM,from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, toabout 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10M to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM toabout 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about75 μM, or from about 75 μM to about 100 μM.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure exhibits increased production in amammalian host cell, compared to the production in the same mammalianhost cell of a control multimeric polypeptide comprising a wild-type4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acidsequence depicted in FIG. 36A or as set forth in SEQ ID NO:213). Forexample, in some cases, a variant 4-1BBL polypeptide present in amultimeric polypeptide, when expressed in a mammalian host cell, isproduced in an amount that is from 25% higher to about 50% higher, fromabout 50% higher to about 75% higher, from about 75% higher to about2-fold higher, from about 2-fold higher to about 5-fold higher, fromabout 5-fold higher to about 10-fold higher, from about 10-fold higherto about 20-fold higher, from about 20-fold higher to about 30-foldhigher, from about 30-fold higher to about 40-fold higher, from about40-fold higher to about 50-fold higher, from about 50-fold higher toabout 75-fold higher, from about 75-fold higher to about 100-foldhigher, or more than 100-fold higher, than the amount of a controlmultimeric polypeptide comprising a wild-type 4-1BBL polypeptide (e.g.,a 4-1BBL polypeptide comprising the amino acid sequence depicted in FIG.36A or as set forth in SEQ ID NO:213) produced in the same mammalianhost cell.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide is produced in a mammalian host cell in an amount of fromabout 50 mg/L to about 75 mg/L, from about 75 mg/L to about 100 mg/L,from about 100 mg/L to about 150 mg/L, from about 150 mg/L to about 200mg/L, from about 200 mg/L to about 250 mg/L, from about 250 mg/L toabout 500 mg/L, or more than 500 mg/L. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide is produced in amammalian host cell in an amount of from about 10 mg/L to about 15 mg/L,from about 15 mg/L to about 20 mg/L, from about 20 mg/L to about 25mg/L, from about 25 mg/L to about 30 mg/L, from about 35 mg/L to about40 mg/L, from about 40 mg/L to about 45 mg/L, or from about 45 mg/L toabout 50 mg/L.

A variant 4-1BBL polypeptide present in a multimeric polypeptide canhave a single amino acid substitution relative to a wild-type 4-1BBLpolypeptide (e.g., a 4-1BBL polypeptide comprising the amino acidsequence depicted in FIG. 36A or as set forth in SEQ ID NO:213). In somecases, a variant 4-1BBL polypeptide present in a multimeric polypeptideof the present disclosure has from 2 to 10 amino acid substitutionsrelative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptidecomprising the amino acid sequence depicted in FIG. 36A or as set forthin SEQ ID NO:213). In some cases, a variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure has 2 amino acidsubstitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBLpolypeptide comprising the amino acid sequence depicted in FIG. 36A oras set forth in SEQ ID NO:213). In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure has 3 amino acid substitutions relative to a wild-type 4-1BBLpolypeptide (e.g., a 4-1BBL polypeptide comprising the amino acidsequence depicted in FIG. 36A or as set forth in SEQ ID NO:213). In somecases, a variant 4-1BBL polypeptide present in a multimeric polypeptideof the present disclosure has 4 amino acid substitutions relative to awild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising theamino acid sequence depicted in FIG. 36A or as set forth in SEQ IDNO:213). In some cases, a variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure has 5 amino acidsubstitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBLpolypeptide comprising the amino acid sequence depicted in FIG. 36A oras set forth in SEQ ID NO:213). In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure has 6 amino acid substitutions relative to a wild-type 4-1BBLpolypeptide (e.g., a 4-1BBL polypeptide comprising the amino acidsequence depicted in FIG. 36A or as set forth in SEQ ID NO:213). In somecases, a variant 4-1BBL polypeptide of the present disclosure has 7amino acid substitutions relative to a wild-type 4-1BBL polypeptide(e.g., a 4-1BBL polypeptide comprising the amino acid sequence depictedin FIG. 36A or as set forth in SEQ ID NO:213). In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure has 8 amino acid substitutions relative to a wild-type 4-1BBLpolypeptide (e.g., a 4-1BBL polypeptide comprising the amino acidsequence depicted in FIG. 36A or as set forth in SEQ ID NO:213). In somecases, a variant 4-1BBL polypeptide present in a multimeric polypeptideof the present disclosure has 9 amino acid substitutions relative to awild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising theamino acid sequence depicted in FIG. 36A or as set forth in SEQ IDNO:213). In some cases, a variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure has 10 amino acidsubstitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBLpolypeptide comprising the amino acid sequence depicted in FIG. 36A oras set forth in SEQ ID NO:213).

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure has from 11 to 50 amino acidsubstitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBLpolypeptide comprising the amino acid sequence depicted in FIG. 36A oras set forth in SEQ ID NO:213). For example, in some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure has from 11 to 15, from 15 to 20, from 20 to 25, from 25 to30, from 30 to 35, from 35 to 40, from 40 to 45, or from 45 to 50, aminoacid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a4-1BBL polypeptide comprising the amino acid sequence depicted in FIG.36A or as set forth in SEQ ID NO:213).

Suitable variant 4-1BBL polypeptides that can be included in amultimeric polypeptide of the present disclosure include those describedabove.

4-1BBL with K127 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where amino acid 127 (indicated by an “x”) is an amino acid other than alysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 47 is an amino acid other than a lysine, e.g.,where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at K48. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at K48. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at K48. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at K48.

K127+M91 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at M91, where amino acid 91 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 91 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 11 is other than methionine, e.g., where amino acid11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn,Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala;and amino acid 11 is Ala.

K127+F92 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at F92, where amino acid 92 is Gly,Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 92 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 12 is other than phenylalanine, e.g., where aminoacid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 isAla; and amino acid 12 is Ala.

K127+094 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at Q94, where amino acid 94 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 94 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 14 is other than glutamine, e.g., where amino acid 14is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 14 is Ala.

K127+L95 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at L95, where amino acid 95 is Gly,Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 95 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 15 is other than leucine, e.g., where amino acid 15is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 15 is Ala.

K127+V96 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at V96, where amino acid 96 is Gly,Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 96 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 16 is other than a valine, e.g., where amino acid 16is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 16 is Ala.

K127+098 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at Q98, where amino acid 98 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 98 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 18 is other than glutamine, e.g., where amino acid 18is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 18 is Ala.

K127+N99 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at N99, where amino acid 99 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 99 is Ala. In some cases, the variant 4-1BBL polypeptide present ina multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 19 is other than an asparagine, e.g., where aminoacid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 isAla; and amino acid 19 is Ala.

K127+V100 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at V100, where amino acid 100 is Gly,Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 100 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 20 is other than a valine, e.g., where amino acid 20is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 20 is Ala.

K127+L101 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at L101, where amino acid 101 is Gly,Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 101 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 21 is other than leucine, e.g., where amino acid 21is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 21 is Ala.

K127+L102 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at L102, where amino acid 102 is Gly,Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 102 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 22 is other than leucine, e.g., where amino acid 22is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 22 is Ala.

K127+I103 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at 1103, where amino acid 103 is Gly,Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 103 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 23 is other than isoleucine, e.g., where amino acid23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala;and amino acid 23 is Ala.

K127+D104 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at D104, where amino acid 104 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 104 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; andii) amino acid 24 is other than aspartic acid, e.g., where amino acid 24is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu. In some cases, amino acid 47 is Ala; andamino acid 24 is Ala.

K127+G105 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at G105, where amino acid 105 is Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 105 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 25 is other than glycine, e.g., where amino acid 25is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 25 is Ala.

K127+P106 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at P106, where amino acid 106 is Gly,Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 106 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 26 is other than proline, e.g., where amino acid 26is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 26 is Ala.

K127+L107 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at L107, where amino acid 107 is Gly,Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 107 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 27 is other than leucine, e.g., where amino acid 27is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 27 is Ala.

K127+S108 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at S108, where amino acid 108 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 108 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 28 is other than serine, e.g., where amino acid 28 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 28 is Ala.

K127+W109 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at W109, where amino acid 109 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 109 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 29 is other than tryptophan, e.g., where amino acid29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala;and amino acid 29 is Ala.

K127+Y110 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at Y110, where amino acid 110 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 110 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 30 is other than tyrosine, e.g., where amino acid 30is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 30 is Ala.

K127+S111 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at S111, where amino acid 111 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 111 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 31 is other than serine, e.g., where amino acid 31 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 31 is Ala.

K127+D112 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at D112, where amino acid 112 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 112 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; andii) amino acid 32 is other than aspartic acid, e.g., where amino acid 32is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu. In some cases, amino acid 47 is Ala; andamino acid 32 is Ala.

K127+P113 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at P113, where amino acid 113 is Gly,Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 113 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 33 is other than proline, e.g., where amino acid 33is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 33 is Ala.

K127+G114 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at G114, where amino acid 114 is Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 114 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 34 is other than glycine, e.g., where amino acid 34is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 34 is Ala.

K127+L115 Substitutions

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36B,where: i) amino acid 127 (indicated by an “x”) is an amino acid otherthan a lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) an amino acid substitution at L115, where amino acid 115 is Gly,Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys,Arg, His, Asp, or Glu. In some cases, amino acid 127 is Ala; and aminoacid 115 is Ala. In some cases, the variant 4-1BBL polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, where: i) amino acid 47 is an amino acid otherthan a lysine, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu;and ii) amino acid 35 is other than leucine, e.g., where amino acid 35is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,Lys, Arg, His, Asp, or Glu. In some cases, amino acid 47 is Ala; andamino acid 35 is Ala.

4-1BBL with Q227 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36Dwhere amino acid 227 (indicated by an “x”) is an amino acid other than aglutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 147 is other than glutamine, e.g., where aminoacid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Q148. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Q148. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at Q148. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at Q148.

4-1BBL with M91 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36E,where amino acid 91 (indicated by an “x”) is an amino acid other than amethionine, e.g., where amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 11 is other than a methionine, e.g., whereamino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at M12. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at M12. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at M12. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at M12.

4-1BBL with F92 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36F,where amino acid 92 (indicated by an “x”) is an amino acid other than aphenylalanine, e.g., where amino acid 92 is Gly, Ala, Val, Leu, Ile,Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 12 is other than a phenylalanine, e.g., whereamino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at F13. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at F13. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at F13. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at F13.

4-1BBL with Q94 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36G,where amino acid 94 (indicated by an “x”) is an amino acid other than aglutamine, e.g., where amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 14 is other than a glutamine, e.g., where aminoacid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Q15. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at Q15. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Q15. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Q15.

4-1BBL with L95 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36H,where amino acid 95 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 15 is other than a leucine, e.g., where aminoacid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L16. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L16. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L16. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L16.

4-1BBL with V96 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36I,where amino acid 96 (indicated by an “x”) is an amino acid other than avaline, e.g., where amino acid 96 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 16 is other than a valine, e.g., where aminoacid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at V17. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at V17. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V17. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V17.

4-1BBL with Q98 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36J,where amino acid 98 (indicated by an “x”) is an amino acid other than aglutamine, e.g., where amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 18 is other than a glutamine, e.g., where aminoacid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Q19. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at Q19. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Q19. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Q19.

4-1BBL with N99 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36K,where amino acid 99 (indicated by an “x”) is an amino acid other than anasparagine, e.g., where amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 19 is other than an asparagine, e.g., whereamino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at N20. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at N20. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at N20. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at N20.

4-1BBL with V100 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36L,where amino acid 100 (indicated by an “x”) is an amino acid other than avaline, e.g., where amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 20 is other than a valine, e.g., where aminoacid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at V21. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at V21. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V21. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V21.

4-1BBL with L101 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36M,where amino acid 101 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 21 is other than a leucine, e.g., where aminoacid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L22. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L22. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L22. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L22.

4-1BBL with L102 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36N,where amino acid 102 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 22 is other than a leucine, e.g., where aminoacid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L23. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L23. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L23. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L23.

4-1BBL with 1103 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36O,where amino acid 103 (indicated by an “x”) is an amino acid other thanan isoleucine, e.g., where amino acid 103 is Gly, Ala, Val, Leu, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 23 is other than an isoleucine, e.g., whereamino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at 124. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at 124. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at 124. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at 124.

4-1BBL with D104 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36P,where amino acid 104 (indicated by an “x”) is an amino acid other thanan aspartic acid, e.g., where amino acid 104 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 24 is other than an aspartic acid, e.g., whereamino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at D25. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at D25. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at D25. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at D25.

4-1BBL with G105 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36Q,where amino acid 105 (indicated by an “x”) is an amino acid other than aglycine, e.g., where amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 25 is other than a glycine, e.g., where aminoacid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G26. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G26. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G26. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G26.

4-1BBL with P106 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36R,where amino acid 106 (indicated by an “x”) is an amino acid other than aproline, e.g., where amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 26 is other than a proline, e.g., where aminoacid 26 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at P27. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at P27. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at P27. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at P27.

4-1BBL with L107 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 365 ,where amino acid 107 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 107 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 27 is other than a leucine, e.g., where aminoacid 27 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L28. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L28. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L28. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L28.

4-1BBL with S108 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36T,where amino acid 108 (indicated by an “x”) is an amino acid other than aserine, e.g., where amino acid 108 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 28 is other than a serine, e.g., where aminoacid 28 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S29. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at S29. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at S29. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at S29.

4-1BBL with W109 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36U,where amino acid 109 (indicated by an “x”) is an amino acid other than atryptophan, e.g., where amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 29 is other than a tryptophan, e.g., whereamino acid 29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at W30. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at W30. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at W30. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at W30.

4-1BBL with Y110 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36V,where amino acid 110 (indicated by an “x”) is an amino acid other than atyrosine, e.g., where amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 30 is other than a tyrosine, e.g., where aminoacid 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Y31. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at Y31. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Y31. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Y31.

4-1BBL with S111 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36W,where amino acid 111 (indicated by an “x”) is an amino acid other than aserine, e.g., where amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 31 is other than a serine, e.g., where aminoacid 31 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S32. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at S32. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at S32. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at S32.

4-1BBL with D112 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36X,where amino acid 112 (indicated by an “x”) is an amino acid other thanan aspartic acid, e.g., where amino acid 112 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 32 is other than an aspartic acid, e.g., whereamino acid 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at D33. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at D33. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at D33. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at D33.

4-1BBL with P113 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36Y,where amino acid 113 (indicated by an “x”) is an amino acid other than aproline, e.g., where amino acid 113 is Gly, Ala, Val, Leu, Ile, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 33 is other than a proline, e.g., where aminoacid 33 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at P34. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at P34. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at P34. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at P34.

4-1BBL with G114 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36Z,where amino acid 114 (indicated by an “x”) is an amino acid other than aglycine, e.g., where amino acid 114 is Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 34 is other than a glycine, e.g., where aminoacid 34 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G35. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G35. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G35. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G35.

4-1BBL with L115 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36AA,where amino acid 115 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 115 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 35 is other than a leucine, e.g., where aminoacid 35 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L36. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L36. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L36. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L36.

4-1BBL with G117 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36BB,where amino acid 117 (indicated by an “x”) is an amino acid other than aglycine, e.g., where amino acid 117 is Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 37 is other than a glycine, e.g., where aminoacid 37 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G38. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G38. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G38. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G38.

4-1BBL with V118 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36CC,where amino acid 118 (indicated by an “x”) is an amino acid other than avaline, e.g., where amino acid 118 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 38 is other than a valine, e.g., where aminoacid 38 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at V39. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at V39. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V39. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V39.

4-1BBL with S119 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36DD,where amino acid 119 (indicated by an “x”) is an amino acid other than aserine, e.g., where amino acid 119 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 39 is other than a serine, e.g., where aminoacid 39 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S40. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at S40. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at S40. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at S40.

4-1BBL with L120 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36EE,where amino acid 120 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 120 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 40 is other than a leucine, e.g., where aminoacid 40 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L41. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L41. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L41. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L41.

4-1BBL with T121 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36FF,where amino acid 121 (indicated by an “x”) is an amino acid other than athreonine, e.g., where amino acid 121 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 41 is other than a threonine, e.g., where aminoacid 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at T42. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at T42. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at T42. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at T42.

4-1BBL with G122 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36GG,where amino acid 122 (indicated by an “x”) is an amino acid other than aglycine, e.g., where amino acid 122 is Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 42 is other than a glycine, e.g., where aminoacid 42 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn,Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G43. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G43. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G43. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G43.

4-1BBL with G123 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36HH,where amino acid 123 (indicated by an “x”) is an amino acid other than aglycine, e.g., where amino acid 123 is Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 43 is other than a glycine, e.g., where aminoacid 43 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn,Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G44. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G44. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G44. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G44.

4-1BBL with L124 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36II,where amino acid 124 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 124 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 44 is other than a leucine, e.g., where aminoacid 44 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L45. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L45. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L45. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L45.

4-1BBL with S125 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36JJ,where amino acid 125 (indicated by an “x”) is an amino acid other than aserine, e.g., where amino acid 125 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 45 is other than a serine, e.g., where aminoacid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S46. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at S46. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at S46. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at S46.

4-1BBL with Y126 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36KK,where amino acid 126 (indicated by an “x”) is an amino acid other than atyrosine, e.g., where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 46 is other than a tyrosine, e.g., where aminoacid 46 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Y47. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at Y47. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Y47. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Y47.

4-1BBL with E128 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36LL,where amino acid 128 (indicated by an “x”) is an amino acid other than aglutamic acid, e.g., where amino acid 128 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 48 is other than a glutamic acid, e.g., whereamino acid 48 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at E49. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at E49. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at E49. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at E49.

4-1BBL with D129 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36MM,where amino acid 129 (indicated by an “x”) is an amino acid other thanan aspartic acid, e.g., where amino acid 129 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 49 is other than an aspartic acid, e.g., whereamino acid 49 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at D50. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at D50. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at D50. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at D50.

4-1BBL with T130 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36NN,where amino acid 130 (indicated by an “x”) is an amino acid other than athreonine, e.g., where amino acid 130 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 50 is other than a threonine, e.g., where aminoacid 50 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at T51. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at T51. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at T51. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at T51.

4-1BBL with K131 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 3600, where amino acid 131 (indicated by an “x”) is an amino acid other thana lysine, e.g., where amino acid 131 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 51 is other than a lysine, e.g., where aminoacid 51 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at K52. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at K52. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at K52. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at K52.

4-1BBL with E132 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36PP,where amino acid 132 (indicated by an “x”) is an amino acid other than aglutamic acid, e.g., where amino acid 132 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 52 is other than a glutamic acid, e.g., whereamino acid 52 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at E53. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at E53. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at E53. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at E53.

4-1BBL with F144 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36QQ,where amino acid 144 (indicated by an “x”) is an amino acid other than aphenylalanine, e.g., where amino acid 144 is Gly, Ala, Val, Leu, Ile,Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 64 is other than a phenylalanine, e.g., whereamino acid 64 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at F65. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at F65. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at F65. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at F65.

4-1BBL with F145 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36RR,where amino acid 145 (indicated by an “x”) is an amino acid other than aphenylalanine, e.g., where amino acid 145 is Gly, Ala, Val, Leu, Ile,Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 65 is other than a phenylalanine, e.g., whereamino acid 65 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at F66. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at F66. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at F66. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at F66.

4-1BBL with Q146 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36SS,where amino acid 146 (indicated by an “x”) is an amino acid other than aglutamine, e.g., where amino acid 146 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 66 is other than a glutamine, e.g., where aminoacid 66 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at Q67. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at Q67. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Q67. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Q67.

4-1BBL with L147Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36TT,where amino acid 147 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 147 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 67 is other than a leucine, e.g., where aminoacid 67 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L68. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L68. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L68. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L68.

4-1BBL with E148 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36UU,where amino acid 148 (indicated by an “x”) is an amino acid other than aglutamic acid, e.g., where amino acid 148 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 68 is other than a glutamic acid, e.g., whereamino acid 68 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at E69. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at E69. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at E69. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at E69.

4-1BBL with L149 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36VV,where amino acid 149 (indicated by an “x”) is an amino acid other than aleucine, e.g., where amino acid 149 is Gly, Ala, Val, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 69 is other than a leucine, e.g., where aminoacid 69 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L70. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at L70. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L70. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L70.

4-1BBL with R150 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36WW,where amino acid 150 (indicated by an “x”) is an amino acid other thanan arginine, e.g., where amino acid 150 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 70 is other than an arginine, e.g., where aminoacid 70 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at R71. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at R71. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at R71. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at R71.

4-1BBL with R1S1 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36XX,where amino acid 151 (indicated by an “x”) is an amino acid other thanan arginine, e.g., where amino acid 151 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu. Insome cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 71 is is other than an arginine, e.g., whereamino acid 71 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at R72. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at R72. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at R72. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at R72.

4-1BBL with V152 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36YY,where amino acid 152 (indicated by an “x”) is an amino acid other than avaline, e.g., where amino acid 152 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 72 is other than a valine, e.g., where aminoacid 72 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at V73. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at V73. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V73. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V73.

4-1BBL with V153 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 36ZZ,where amino acid 153 (indicated by an “x”) is an amino acid other than avaline, e.g., where amino acid 153 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In somecases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 73 is other than a valine, e.g., where aminoacid 73 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at V74. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at V74. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V74. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V74.

4-1BBL with G155 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36AAA, where amino acid 155 (indicated by an “x”) is an amino acid otherthan a glycine, e.g., where amino acid 155 is Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 75 is other than a glycine, e.g., where aminoacid 75 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G76. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G76. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G76. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G76.

4-1BBL with E156 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36BBB, where amino acid 156 (indicated by an “x”) is an amino acid otherthan a glutamic acid, e.g., where amino acid 156 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, orAsp. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 76 is other than a glutamic acid,e.g., where amino acid 76 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at E77. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at E77. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at E77. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at E77.

4-1BBL with G157 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36CCC, where amino acid 157 (indicated by an “x”) is an amino acid otherthan a glycine, e.g., where amino acid 157 is Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 77 is other than a glycine, e.g., where aminoacid 77 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at G78. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at G78. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G78. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G78.

4-1BBL with S158 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36DDD, where amino acid 158 (indicated by an “x”) is an amino acid otherthan a serine, e.g., where amino acid 158 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 78 is other than a serine, e.g., where aminoacid 78 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S79. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214,with an amino acid substitution at S79. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at S79. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at S79.

4-1BBL with D184 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36EEE, where amino acid 184 (indicated by an “x”) is an amino acid otherthan an aspartic acid, e.g., where amino acid 184 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, orGlu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 104 is other than an aspartic acid,e.g., where amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at D105. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at D105. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at D105. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at D105.

4-1BBL with L185 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36FFF, where amino acid 185 (indicated by an “x”) is an amino acid otherthan a leucine, e.g., where amino acid 185 is Gly, Ala, Val, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 105 is other than a leucine, e.g., where aminoacid 105 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L106. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at L106. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at L106. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at L106.

4-1BBL with P186 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36GGG, where amino acid 186 (indicated by an “x”) is an amino acid otherthan a proline, e.g., where amino acid 186 is Gly, Ala, Val, Leu, Ile,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 106 is other than a proline, e.g., where aminoacid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at P107. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at P107. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at P107. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at P107.

4-1BBL with P187Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36HHH, where amino acid 187 (indicated by an “x”) is an amino acid otherthan a proline, e.g., where amino acid 187 is Gly, Ala, Val, Leu, Ile,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 107 is other than a proline, e.g., where aminoacid 107 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at P108. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at P108. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at P108. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at P108.

4-1BBL with S189 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36III, where amino acid 189 (indicated by an “x”) is an amino acid otherthan a serine, e.g., where amino acid 189 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 109 is other than a serine, e.g., where aminoacid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at 5110. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at 5110. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at 5110. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at 5110.

4-1BBL with S190 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36JJJ, where amino acid 190 (indicated by an “x”) is an amino acid otherthan a serine, e.g., where amino acid 190 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 110 is other than a serine, e.g., where aminoacid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S111. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at S111. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at S111. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at S111.

4-1BBL with E191 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36KKK, where amino acid 191 (indicated by an “x”) is an amino acid otherthan a glutamic acid, e.g., where amino acid 191 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, orAsp. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 111 is other than a glutamic acid,e.g., where amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at E112. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence set forth in SEQ IDNO:214, with an amino acid substitution at E112. In some cases, avariant 4-1BBL polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:215, with anamino acid substitution at E112. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:215,with an amino acid substitution at E112.

4-1BBL with R193 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36LLL, where amino acid 193 (indicated by an “x”) is an amino acid otherthan an arginine, e.g., where amino acid 193 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 113 is other than arginine, e.g., where aminoacid 113 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at R114. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at R114. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at R114. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at R114.

4-1BBL with N194 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36MMM, where amino acid 194 (indicated by an “x”) is an amino acid otherthan a asparagine, e.g., where amino acid 194 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, orGlu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 114 is other than a asparagine, e.g.,where amino acid 114 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at N115. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at N115. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at N115. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at N115.

4-1BBL with S195 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36NNN, where amino acid 195 (indicated by an “x”) is an amino acid otherthan a serine, e.g., where amino acid 195 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 115 is other than a serine, e.g., where aminoacid 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at S116. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at S116. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at S116. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at S116.

4-1BBL with F197 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36OOO, where amino acid 197 (indicated by an “x”) is an amino acid otherthan a phenylalanine, e.g., where amino acid 197 is Gly, Ala, Val, Leu,Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, orGlu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 117 is other than a phenylalanine,e.g., where amino acid 117 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at F118. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at F118. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at F118. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at F118.

4-1BBL with Q210 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36PPP, where amino acid 210 (indicated by an “x”) is an amino acid otherthan a glutamine, e.g., where amino acid 210 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 130 is other than a glutamine, e.g., whereamino acid 130 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at Q131. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at Q131. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Q131. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Q131.

4-1BBL with R211 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36QQQ, where amino acid 211 (indicated by an “x”) is an amino acid otherthan an arginine, e.g., where amino acid 211 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 131 is other than an arginine, e.g., whereamino acid 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at R132. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at R132. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at R132. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at R132.

4-1BBL with L212 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36RRR, where amino acid 212 (indicated by an “x”) is an amino acid otherthan a leucine, e.g., where amino acid 212 is Gly, Ala, Val, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 132 is other than a leucine, e.g., where aminoacid 132 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at L133. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at L133. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L133. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L133.

4-1BBL with G213 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36SSS, where amino acid 213 (indicated by an “x”) is an amino acid otherthan a glycine, e.g., where amino acid 213 is Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 133 is other than a glycine, e.g., where aminoacid 133 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at G134. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at G134. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G134. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G134.

4-1BBL with V214 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36TTT, where amino acid 214 (indicated by an “x”) is an amino acid otherthan a valine, e.g., where amino acid 214 is Gly, Ala, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 134 is other than a valine, e.g., where aminoacid 134 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at V135. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at V135. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V135. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V135.

4-1BBL with H215 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36UUU, where amino acid 215 (indicated by an “x”) is an amino acid otherthan a histidine, e.g., where amino acid 215 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 135 is other than a histidine, e.g., whereamino acid 135 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at H136. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at H136. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at H136. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at H136.

4-1BBL with L216 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36VVV, where amino acid 216 (indicated by an “x”) is an amino acid otherthan a leucine, e.g., where amino acid 216 is Gly, Ala, Val, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 136 is other than a leucine, e.g., where aminoacid 136 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at L137. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at L137. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L137. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L137.

4-1BBL with H217 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36WWW, where amino acid 217 (indicated by an “x”) is an amino acid otherthan a histidine, e.g., where amino acid 217 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 137 is other than a histidine, e.g., whereamino acid 137 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at H138. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at H138. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at H138. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at H138.

4-1BBL with T218 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36XXX, where amino acid 218 (indicated by an “x”) is an amino acid otherthan a threonine, e.g., where amino acid 218 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 138 is other than a threonine, e.g., whereamino acid 138 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys,Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at T139. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at T139. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at T139. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at T139.

4-1BBL with E219 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36YYY, where amino acid 219 (indicated by an “x”) is an amino acid otherthan a glutamic acid, e.g., where amino acid 219 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, orAsp. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 139 is other than a glutamic acid,e.g., where amino acid 139 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Asp.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at E140. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at E140. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at E140. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at E140.

4-1BBL with R221 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36ZZZ, where amino acid 221 (indicated by an “x”) is an amino acid otherthan an arginine, e.g., where amino acid 221 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:213, where amino acid 141 is other than an arginine, e.g., whereamino acid 141 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at R142. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at R142. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at R142. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at R142.

4-1BBL with R223 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36AAAA, where amino acid 223 (indicated by an “x”) is an amino acidother than an arginine, e.g., where amino acid 223 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His,Asp, or Glu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 143 is other than an arginine, e.g.,where amino acid 143 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at R144. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at R144. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at R144. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at R144.

4-1BBL with H224 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36BBBB, where amino acid 224 (indicated by an “x”) is an amino acidother than a histidine, e.g., where amino acid 224 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg,Asp, or Glu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 144 is other than a histidine, e.g.,where amino acid 144 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at H145. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at H145. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at H145. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at H145.

4-1BBL with W226 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36CCCC, where amino acid 226 (indicated by an “x”) is an amino acidother than a tryptophan, e.g., where amino acid 226 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 146 is other than a tryptophan, e.g.,where amino acid 146 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser,Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at W147. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at W147. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at W147. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at W147.

4-1BBL with L228 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36DDDD, where amino acid 228 (indicated by an “x”) is an amino acidother than a leucine, e.g., where amino acid 228 is Gly, Ala, Val, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, orGlu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 148 is other than a leucine, e.g.,where amino acid 148 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser,Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at L149. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at L149. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at L149. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at L149.

4-1BBL with T229 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36EEEE, where amino acid 229 (indicated by an “x”) is an amino acidother than a threonine, e.g., where amino acid 229 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 149 is other than a threonine, e.g.,where amino acid 149 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at T150. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at T150. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at T150. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at T150.

4-1BBL with Q230 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36FFFF, where amino acid 230 (indicated by an “x”) is an amino acidother than a glutamine, e.g., where amino acid 230 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His,Asp, or Glu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 150 is other than a glutamine, e.g.,where amino acid 150 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at Q151. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at Q151. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at Q151. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at Q151.

4-1BBL with G231 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36GGGG, where amino acid 231 (indicated by an “x”) is an amino acidother than a glycine, e.g., where amino acid 231 is Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, orGlu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 151 is other than a glycine, e.g.,where amino acid 151 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at G152. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at G152. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at G152. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at G152.

4-1BBL with T233 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36HHHH, where amino acid 233 (indicated by an “x”) is an amino acidother than a threonine, e.g., where amino acid 233 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 153 is other than a threonine, e.g.,where amino acid 153 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at T154. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at T154. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at T154. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at T154.

4-1BBL with V234 Substitution

In some cases, the variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG.36IIII, where amino acid 234 (indicated by an “x”) is an amino acidother than a valine, e.g., where amino acid 234 is Gly, Ala, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, orGlu. In some cases, the variant 4-1BBL polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, where amino acid 154 is other than a valine, e.g.,where amino acid 154 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, a variant 4-1BBL polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:214 with an amino acid substitution at V155. In some cases, a variant4-1BBL polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence set forth in SEQ ID NO:214with an amino acid substitution at V155. In some cases, a variant 4-1BBLpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:215, with an aminoacid substitution at V155. In some cases, a variant 4-1BBL polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence set forth in SEQ ID NO:215, with an amino acidsubstitution at V155.

Exemplary Multimeric Polypeptides Comprising 4-1BBL ImmunomodulatoryPolypeptide

Exemplary multimeric polypeptides that are suitable for use in a methodof the present disclosure are described below.

K127

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; ii) a β2M polypeptide; and iii) a variant4-1BBL polypeptide comprising an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 36B, where aminoacid 127 (indicated by an “x”) is an amino acid other than a lysine,e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; or a variant4-1BBL polypeptide of the present disclosure comprising an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, with an amino acid substitution at K47, e.g., whereamino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus: i) a Class I MHCheavy chain; and ii) an Fc polypeptide. In some cases, a multimericpolypeptide of the present disclosure comprises: a) a first polypeptidecomprising, in order from N-terminus to C-terminus: i) an epitope; andii) a β2M polypeptide; and b) a second polypeptide comprising, in orderfrom N-terminus to C-terminus: i) a variant 4-1BBL polypeptidecomprising an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 36B, where amino acid 127 (indicated byan “x”) is an amino acid other than a lysine, e.g., where amino acid 127is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Arg, His, Asp, or Glu; or a variant 4-1BBL polypeptide of thepresent disclosure comprising an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:213, with anamino acid substitution at K47, e.g., where amino acid 47 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg,His, Asp, or Glu; ii) a Class I MHC heavy chain; and iii) an Fcpolypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus: i) an epitope; ii) a β2M polypeptide; iii) afirst variant 4-1BBL polypeptide of the present disclosure; iv) a secondvariant 4-1BBL polypeptide of the present disclosure; and v) a thirdvariant 4-1BBL polypeptide of the present disclosure; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus: i) aClass I MHC heavy chain; and ii) an Fc polypeptide. In some cases, eachof the first, second, and third variant 4-1BBL polypeptides comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 36B, where amino acid 127 (indicated by an “x”) is anamino acid other than a lysine, e.g., where amino acid 127 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg,His, Asp, or Glu; e.g., each of the first, second, and third variant4-1BBL polypeptides comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:213, with anamino acid substitution at K47, e.g., where amino acid 47 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg,His, Asp, or Glu. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus: i) an epitope; and ii) a β2M polypeptide; andb) a second polypeptide comprising, in order from N-terminus toC-terminus: i) a first variant 4-1BBL polypeptide of the presentdisclosure; ii) a second variant 4-1BBL polypeptide of the presentdisclosure; and iii) a third variant 4-1BBL polypeptide of the presentdisclosure; iv) a Class I MHC heavy chain; and v) an Fc polypeptide. Insome cases, each of the first, second, and third variant 4-1BBLpolypeptides comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 36B, where amino acid 127(indicated by an “x”) is an amino acid other than a lysine, e.g., whereamino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; e.g., each of the first,second, and third variant 4-1BBL polypeptides comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, with an amino acid substitution at K47, e.g., whereamino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In some cases, the variant4-1BBL polypeptide comprises a K127 substitution and an M91 substitution(based on the numbering depicted in FIG. 36A). In some cases, thevariant 4-1BBL polypeptide comprises a K127 substitution and an F92substitution (based on the numbering depicted in FIG. 36A). In somecases, the variant 4-1BBL polypeptide comprises a K127 substitution anda Q94 substitution (based on the numbering depicted in FIG. 36A). Insome cases, the variant 4-1BBL polypeptide comprises a K127 substitutionand an L95 substitution (based on the numbering depicted in FIG. 36A).In some cases, the variant 4-1BBL polypeptide comprises a K127substitution and a V96 substitution (based on the numbering depicted inFIG. 36A). In some cases, the variant 4-1BBL polypeptide comprises aK127 substitution and a Q98 substitution (based on the numberingdepicted in FIG. 36A). In some cases, the variant 4-1BBL polypeptidecomprises a K127 substitution and an N99 substitution (based on thenumbering depicted in FIG. 36A). In some cases, the variant 4-1BBLpolypeptide comprises a K127 substitution and a V100 substitution (basedon the numbering depicted in FIG. 36A). In some cases, the variant4-1BBL polypeptide comprises a K127 substitution and an L101substitution (based on the numbering depicted in FIG. 36A). In somecases, the variant 4-1BBL polypeptide comprises a K127 substitution andan L102 substitution (based on the numbering depicted in FIG. 36A). Insome cases, the variant 4-1BBL polypeptide comprises a K127 substitutionand an 1103 substitution (based on the numbering depicted in FIG. 36A).In some cases, the variant 4-1BBL polypeptide comprises a K127substitution and a D104 substitution (based on the numbering depicted inFIG. 36A). In some cases, the variant 4-1BBL polypeptide comprises aK127 substitution and a G105 substitution (based on the numberingdepicted in FIG. 36A). In some cases, the variant 4-1BBL polypeptidecomprises a K127 substitution and a P106 substitution (based on thenumbering depicted in FIG. 36A). In some cases, the variant 4-1BBLpolypeptide comprises a K127 substitution and an L107 substitution(based on the numbering depicted in FIG. 36A). In some cases, thevariant 4-1BBL polypeptide comprises a K127 substitution and an S108substitution (based on the numbering depicted in FIG. 36A). In somecases, the variant 4-1BBL polypeptide comprises a K127 substitution anda W109 substitution (based on the numbering depicted in FIG. 36A). Insome cases, the variant 4-1BBL polypeptide comprises a K127 substitutionand a Y110 substitution (based on the numbering depicted in FIG. 36A).In some cases, the variant 4-1BBL polypeptide comprises a K127substitution and an S111 substitution (based on the numbering depictedin FIG. 36A). In some cases, the variant 4-1BBL polypeptide comprises aK127 substitution and a D112 substitution (based on the numberingdepicted in FIG. 36A). In some cases, the variant 4-1BBL polypeptidecomprises a K127 substitution and a P113 substitution (based on thenumbering depicted in FIG. 36A). In some cases, the variant 4-1BBLpolypeptide comprises a K127 substitution and a G114 substitution (basedon the numbering depicted in FIG. 36A). In some cases, the variant4-1BBL polypeptide comprises a K127 substitution and an L115substitution (based on the numbering depicted in FIG. 36A).

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; and ii) a β2M polypeptide; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus: i) afirst variant 4-1BBL polypeptide of the present disclosure; ii) alinker; iii) a second variant 4-1BBL polypeptide of the presentdisclosure; iv) a linker; v) a third variant 4-1BBL polypeptide of thepresent disclosure; vi) a Class I MHC heavy chain; and vii) an Fcpolypeptide. In some cases, each of the first, second, and third variant4-1BBL polypeptides comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 36B, where aminoacid 127 (indicated by an “x”) is an amino acid other than a lysine,e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu; e.g., each ofthe first, second, and third variant 4-1BBL polypeptides comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO:213, with an amino acid substitution at K47,e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In some cases,the linker comprises a (GSSSS)n (SEQ ID NO:131) sequence, where n is 1,2, 3, 4, or 5. In some cases, n is 4. In some cases, n is 5. In somecases, the variant 4-1BBL polypeptide comprises a K127 substitution andan M91 substitution (based on the numbering depicted in FIG. 36A). Insome cases, the variant 4-1BBL polypeptide comprises a K127 substitutionand an F92 substitution (based on the numbering depicted in FIG. 36A).In some cases, the variant 4-1BBL polypeptide comprises a K127substitution and a Q94 substitution (based on the numbering depicted inFIG. 36A). In some cases, the variant 4-1BBL polypeptide comprises aK127 substitution and an L95 substitution (based on the numberingdepicted in FIG. 36A). In some cases, the variant 4-1BBL polypeptidecomprises a K127 substitution and a V96 substitution (based on thenumbering depicted in FIG. 36A). In some cases, the variant 4-1BBLpolypeptide comprises a K127 substitution and a Q98 substitution (basedon the numbering depicted in FIG. 36A). In some cases, the variant4-1BBL polypeptide comprises a K127 substitution and an N99 substitution(based on the numbering depicted in FIG. 36A). In some cases, thevariant 4-1BBL polypeptide comprises a K127 substitution and a V100substitution (based on the numbering depicted in FIG. 36A). In somecases, the variant 4-1BBL polypeptide comprises a K127 substitution andan L101 substitution (based on the numbering depicted in FIG. 36A). Insome cases, the variant 4-1BBL polypeptide comprises a K127 substitutionand an L102 substitution (based on the numbering depicted in FIG. 36A).In some cases, the variant 4-1BBL polypeptide comprises a K127substitution and an 1103 substitution (based on the numbering depictedin FIG. 36A). In some cases, the variant 4-1BBL polypeptide comprises aK127 substitution and a D104 substitution (based on the numberingdepicted in FIG. 36A). In some cases, the variant 4-1BBL polypeptidecomprises a K127 substitution and a G105 substitution (based on thenumbering depicted in FIG. 36A). In some cases, the variant 4-1BBLpolypeptide comprises a K127 substitution and a P106 substitution (basedon the numbering depicted in FIG. 36A). In some cases, the variant4-1BBL polypeptide comprises a K127 substitution and an L107substitution (based on the numbering depicted in FIG. 36A). In somecases, the variant 4-1BBL polypeptide comprises a K127 substitution andan S108 substitution (based on the numbering depicted in FIG. 36A). Insome cases, the variant 4-1BBL polypeptide comprises a K127 substitutionand a W109 substitution (based on the numbering depicted in FIG. 36A).In some cases, the variant 4-1BBL polypeptide comprises a K127substitution and a Y110 substitution (based on the numbering depicted inFIG. 36A). In some cases, the variant 4-1BBL polypeptide comprises aK127 substitution and an S111 substitution (based on the numberingdepicted in FIG. 36A). In some cases, the variant 4-1BBL polypeptidecomprises a K127 substitution and a D112 substitution (based on thenumbering depicted in FIG. 36A). In some cases, the variant 4-1BBLpolypeptide comprises a K127 substitution and a P113 substitution (basedon the numbering depicted in FIG. 36A). In some cases, the variant4-1BBL polypeptide comprises a K127 substitution and a G114 substitution(based on the numbering depicted in FIG. 36A). In some cases, thevariant 4-1BBL polypeptide comprises a K127 substitution and an L115substitution (based on the numbering depicted in FIG. 36A).

Q227

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; ii) a β2M polypeptide; and iii) a variant4-1BBL polypeptide comprising an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 36D, where aminoacid 227 (indicated by an “x”) is an amino acid other than a glutamine,e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; or a variant4-1BBL polypeptide of the present disclosure comprising an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence set forthin SEQ ID NO:213, with an amino acid substitution at Q147, e.g., whereamino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus: i) a Class I MHCheavy chain; and ii) an Fc polypeptide. In some cases, a multimericpolypeptide of the present disclosure comprises: a) a first polypeptidecomprising, in order from N-terminus to C-terminus: i) an epitope; andii) a β2M polypeptide; and b) a second polypeptide comprising, in orderfrom N-terminus to C-terminus: i) a variant 4-1BBL polypeptidecomprising an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 36D, where amino acid 227 (indicated byan “x”) is an amino acid other than a glutamine, e.g., where amino acid227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met,Asn, Lys, Arg, His, Asp, or Glu; or a variant 4-1BBL polypeptide of thepresent disclosure comprising an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:213, with anamino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg,His, Asp, or Glu; ii) a Class I MHC heavy chain; and iii) an Fcpolypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus: i) an epitope; ii) a β2M polypeptide; iii) afirst variant 4-1BBL polypeptide of the present disclosure; iv) a secondvariant 4-1BBL polypeptide of the present disclosure; and v) a thirdvariant 4-1BBL polypeptide of the present disclosure; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus: i) aClass I MHC heavy chain; and ii) an Fc polypeptide. In some cases, eachof the first, second, and third variant 4-1BBL polypeptides comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 36D, where amino acid 227 (indicated by an “x”) is anamino acid other than a glutamine, e.g., where amino acid 227 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys,Arg, His, Asp, or Glu; or each of the first, second, and third variant4-1BBL polypeptides comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:213, with anamino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg,His, Asp, or Glu. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus: i) an epitope; and ii) a β2M polypeptide; andb) a second polypeptide comprising, in order from N-terminus toC-terminus: i) a first variant 4-1BBL polypeptide of the presentdisclosure; ii) a second variant 4-1BBL polypeptide of the presentdisclosure; and iii) a third variant 4-1BBL polypeptide of the presentdisclosure; iv) a Class I MHC heavy chain; and v) an Fc polypeptide. Insome cases, each of the first, second, and third variant 4-1BBLpolypeptides comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 36D, where amino acid 227(indicated by an “x”) is an amino acid other than a glutamine, e.g.,where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; or each of thefirst, second, and third variant 4-1BBL polypeptides comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, with an amino acid substitution at Q147, e.g.,where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus: i) an epitope; and ii) a β2M polypeptide; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus: i) afirst variant 4-1BBL polypeptide of the present disclosure; ii) alinker; iii) a second variant 4-1BBL polypeptide of the presentdisclosure; iv) a linker; v) a third variant 4-1BBL polypeptide of thepresent disclosure; vi) a Class I MHC heavy chain; and vii) an Fcpolypeptide. In some cases, each of the first, second, and third variant4-1BBL polypeptides comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 36D, where aminoacid 227 (indicated by an “x”) is an amino acid other than a glutamine,e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr,Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; or each of thefirst, second, and third variant 4-1BBL polypeptides comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:213, with an amino acid substitution at Q147, e.g.,where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, thelinker comprises a (GSSSS)n (SEQ ID NO:131) sequence, where n is 1, 2,3, 4, or 5. In some cases, the linker comprises a (GSSSS)n (SEQ IDNO:131) sequence, where n is 4. In some cases, the linker comprises a(GSSSS)n (SEQ ID NO:131) sequence, where n is 5. In some cases, thelinker comprises a (GGGGS)n sequence, where n is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10. In some cases, the linker comprises a (GGGGS)n sequence, wheren is 2. In some cases, the linker comprises a (GGGGS)n sequence, where nis 3. In some cases, the linker comprises a (GGGGS)n sequence, where nis 4. In some cases, the linker comprises a (GGGGS)n sequence, where nis 5.

Multiple Immunomodulatory Domains

As noted above, in some cases, a multimeric polypeptide comprises two ormore immunomodulatory polypeptides. In some cases, at least one of thetwo or more immunomodulatory polypeptide is a variant immunomodulatorypolypeptide. For example, in the case of an IL-2/synTac, in some cases,at least one of the two or more immunomodulatory polypeptide is avariant IL-2 polypeptide. As another example, in the case of a4-1BBL/synTac, in some cases, at least one of the two or moreimmunomodulatory polypeptide is a variant 4-1BBL polypeptide.

In some cases, a multimeric polypeptide comprises two or more copies ofa variant IL-2 polypeptide of the present disclosure. In some cases, thetwo or more variant IL-2 polypeptides are on the same polypeptide chainof a multimeric polypeptide. In some cases, the two or more variant IL-2polypeptides are on separate polypeptide chains of a multimericpolypeptide.

In some cases, a multimeric polypeptide comprises a firstimmunomodulatory polypeptide, and at least a second immunomodulatorypolypeptide, where the first immunomodulatory polypeptide is a variantIL-2 polypeptide of the present disclosure, and the secondimmunomodulatory polypeptide is not an IL-2 polypeptide. For example, insome cases, the second immunomodulatory polypeptide is a member of thetumor necrosis factor (TNF) superfamily; e.g., a FasL polypeptide, a4-1BBL polypeptide, a CD40 polypeptide, an OX40L polypeptide, a CD30Lpolypeptide, a CD70 polypeptide, etc. In some cases, the secondimmunomodulatory polypeptide of a multimeric polypeptide is a T-cellco-stimulatory polypeptide and is a member of the immunoglobulin (Ig)superfamily; e.g., a CD7 polypeptide, a CD86 polypeptide, an ICAMpolypeptide, etc. In some cases, the second immunomodulatory polypeptideis 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1, CD86, FasL, and PD-L2. Suitableimmunomodulatory polypeptides of a multimeric polypeptide of the presentdisclosure include, e.g., CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA,MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, or HVEM.

Further T cell modulatory domains (MODs) that can be included in amultimeric polypeptide of the present disclosure include naturallyoccurring or synthetic human gene products (protein), affinity reagents(e.g., an antibody, antibody fragment, single chain Fvs, aptamers,nanobody) targeting a human gene product, including, but not limited toall secreted proteins arising from classical and non-classical (e.g.,FGF2, IL1, S100A4) secretion mechanisms, and ecto-domains of all cellsurface proteins anchored by naturally occurring genetically encodedprotein segments (single or multiple membrane spans) orpost-translational modifications such as GPI linkages). Any naturallyoccurring or synthetic affinity reagent (e.g., antibody, antibodyfragment, single chain Fvs, aptamer, nanobody, lectin, etc) targeting acell surface glycan or other post-translational modification (e.g.,sulfation). Examples include, but are not limited to, members of theTNF/TNFR family (OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9,RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG,CD70) or affinity reagents directed at the TNF/TNFR family members;members of the Immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2,B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chains,ICOS, ICOS ligand, HHLA2, butyrophilins, BTLA, B7-H3, B7-H4, CD3, CD79a,CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244,ICAM-1), Leukocyte immunoglobulin like receptors (LILR), killer cellimmunoglobulin like receptors (KIR)), lectin superfamily members,selectins, cytokines/chemokine and cytokine/chemokine receptors, growthfactors and growth factor receptors), adhesion molecules (integrins,fibronectins, cadherins), or ecto-domains of multi-span integralmembrane protein, or affinity reagents directed at the Immunoglobulinsuperfamily and listed gene products. In addition, activehomologs/orthologs of these gene products, including but not limited to,viral sequences (e.g., CMV, EBV), bacterial sequences, fungal sequences,eukaryotic pathogens (e.g., Schistosoma, Plasmodium, Babesia, Eimeria,Theileria, Toxoplasma, Entamoeba, Leishmania, and Trypanosoma), andmammalian-derived coding regions. In addition, a MOD may comprise asmall molecules drug targeting a human gene product.

Additional Polypeptides

A polypeptide chain of a multimeric polypeptide can include one or morepolypeptides in addition to those described above. Suitable additionalpolypeptides include epitope tags and affinity domains. The one or moreadditional polypeptide can be included at the N-terminus of apolypeptide chain of a multimeric polypeptide, at the C-terminus of apolypeptide chain of a multimeric polypeptide, or internally within apolypeptide chain of a multimeric polypeptide.

Epitope Tag

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., YPYDVPDYA (SEQ ID NO:79); FLAG (e.g., DYKDDDDK (SEQ IDNO:80); c-myc (e.g., EQKLISEEDL; SEQ ID NO:81), and the like.

Affinity Domain

Affinity domains include peptide sequences that can interact with abinding partner, e.g., such as one immobilized on a solid support,useful for identification or purification. DNA sequences encodingmultiple consecutive single amino acids, such as histidine, when fusedto the expressed protein, may be used for one-step purification of therecombinant protein by high affinity binding to a resin column, such asnickel sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQID NO:82), HisX6 (HHHHHH) (SEQ ID NO:83), C-myc (EQKLISEEDL) (SEQ IDNO:81), Flag (DYKDDDDK) (SEQ ID NO:80), StrepTag (WSHPQFEK) (SEQ IDNO:84), hemagglutinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:79),glutathione-S-transferase (GST), thioredoxin, cellulose binding domain,RYIRS (SEQ ID NO:85), Phe-His-His-Thr (SEQ ID NO:86), chitin bindingdomain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,WEAAAREACCRECCARA (SEQ ID NO:87), metal binding domains, e.g., zincbinding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin,inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, andmaltose binding protein.

Examples of IL-2/Multimeric Polypeptides

The following are non-limiting embodiments of an IL-2/synTac multimericpolypeptide suitable for use in a treatment method of the presentdisclosure.

In some cases, an IL-2/synTac multimeric polypeptide comprises: a) afirst polypeptide comprising, in order from N-terminus to C-terminus: i)an epitope; ii) a 02-microglobulin (β2M) polypeptide comprising theamino acid sequence depicted in FIG. 34A; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus: i) a variant IL-2polypeptide of the present disclosure; ii) a major histocompatibilitycomplex (MHC) heavy chain polypeptide comprising the amino acid sequencedepicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising one ormore amino acid substitutions selected from N297A, L234A, L235A, L234F,L235E, and P331S. In some cases, the variant IL-2 polypeptide comprisesan H16A and an F42A substitution. In some cases, the IgG1 Fc polypeptidecomprises an N297A substitution. In some cases, the IgG1 Fc polypeptidecomprises an L234A substitution and an L235A substitution. In somecases, the IgG1 Fc polypeptide comprises an L234F substitution and anL235E substitution. In some cases, the IgG1 Fc polypeptide comprises anL234F substitution, an L235E substitution, and a P33IS substitution. Insome cases, the second polypeptide comprises two copies of the variantIL-2 polypeptide. In some cases, the first polypeptide comprises apeptide linker between the epitope and the β2M polypeptide. In somecases, the second polypeptide comprises a peptide linker between one ormore of: a) a first copy of the variant IL-2 polypeptide and a secondcopy of the variant IL-2 polypeptide; b) the variant IL-2 polypeptideand the MHC heavy chain polypeptide; and c) between the MHC heavy chainpolypeptide and the IgG1 Fc polypeptide. In some cases, the peptidelinker is selected from (GGGGS)₃ (SEQ ID NO:207), (GGGGS)₄ (SEQ IDNO:208), and AAAGG (SEQ ID NO:73). In some cases, the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33B. Insome cases, the IgG1 Fc polypeptide comprises the amino acid sequencedepicted in FIG. 33C. In some cases, the IgG1 Fc polypeptide comprisesthe amino acid sequence depicted in FIG. 33D.

In some cases, a multimeric polypeptide comprises: a) a firstpolypeptide comprising, in order from N-terminus to C-terminus: i) anepitope; ii) a 02-microglobulin polypeptide comprising the amino acidsequence depicted in FIG. 34A; and b) a second polypeptide comprising,in order from N-terminus to C-terminus: i) a variant IL-2 polypeptidecomprising the amino acid sequence depicted in FIG. 34B; ii) a majorhistocompatibility complex (MHC) heavy chain polypeptide comprising theamino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fcpolypeptide comprising one or more amino acid substitutions selectedfrom N297A, L234A, L235A, L234F, L235E, and P331S. In some cases, theIgG1 Fc polypeptide comprises an N297A substitution. In some cases, theIgG1 Fc polypeptide comprises an L234A substitution and an L235Asubstitution. In some cases, the IgG1 Fc polypeptide comprises an L234Fsubstitution and an L235E substitution. In some cases, the IgG1 Fcpolypeptide comprises an L234F substitution, an L235E substitution, anda P331S substitution. In some cases, the IgG1 Fc polypeptide comprisesthe amino acid sequence depicted in FIG. 33B. In some cases, the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33C. Insome cases, the IgG1 Fc polypeptide comprises the amino acid sequencedepicted in FIG. 33D. In some cases, in the second polypeptide comprisestwo copies of the variant IL-2 polypeptide. In some cases, the firstpolypeptide comprises a peptide linker between the epitope and the β2Mpolypeptide. In some cases, the second polypeptide comprises a peptidelinker between one or more of: a) a first copy of the variant IL-2polypeptide and a second copy of the variant IL-2 polypeptide; b) thevariant IL-2 polypeptide and the MHC heavy chain polypeptide; and c)between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide. Insome cases, the peptide linker is selected from (GGGGS)₃ (SEQ IDNO:207), (GGGGS)₄ (SEQ ID NO:208), and AAAGG (SEQ ID N0:73).

In some cases, multimeric polypeptide comprises: a) a first polypeptidecomprising, in order from N-terminus to C-terminus: i) an epitopecomprising the amino acid sequence YMLDLQPETT (SEQ ID NO:77); ii) a02-microglobulin polypeptide comprising the amino acid sequence depictedin FIG. 34A; and b) a second polypeptide comprising, in order fromN-terminus to C-terminus: i) a variant IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 34B; ii) a major histocompatibilitycomplex (MHC) heavy chain polypeptide comprising the amino acid sequencedepicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising theamino acid sequence depicted in FIG. 33A, 33B, 33C, or 33D. In somecases, the IgG1 Fc polypeptide comprises the amino acid sequencedepicted in FIG. 33B. In some cases, the IgG1 Fc polypeptide comprisesthe amino acid sequence depicted in FIG. 33C. In some cases, the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33D. Insome cases, the second polypeptide comprises two copies of the variantIL-2 polypeptide. In some cases, the first polypeptide comprises apeptide linker between the epitope and the β2M polypeptide. In somecases, the second polypeptide comprises a peptide linker between one ormore of: a) a first copy of the variant IL-2 polypeptide and a secondcopy of the variant IL-2 polypeptide; b) the variant IL-2 polypeptideand the MHC heavy chain polypeptide; and c) between the MHC heavy chainpolypeptide and the IgG1 Fc polypeptide. In some cases, the peptidelinker is selected from (GGGGS)₃ (SEQ ID NO:207), (GGGGS)₄ (SEQ IDNO:208), and AAAGG (SEQ ID NO:73). In some cases, the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33B. Insome cases, the IgG1 Fc polypeptide comprises the amino acid sequencedepicted in FIG. 33C. In some cases, the IgG1 Fc polypeptide comprisesthe amino acid sequence depicted in FIG. 33D.

In some cases, a multimeric polypeptide comprises: a) a firstpolypeptide comprising the amino acid sequence depicted in FIG. 31 ; andb) a second polypeptide comprising the amino acid sequence depicted inFIG. 22 .

In some cases, a multimeric polypeptide comprises: a) a firstpolypeptide comprising the amino acid sequence depicted in FIG. 31 ; andb) a second polypeptide comprising the amino acid sequence depicted inFIG. 25 .

In some cases, a multimeric polypeptide comprises: a) a firstpolypeptide comprising the amino acid sequence depicted in FIG. 31 ; andab) a second polypeptide comprising the amino acid sequence depicted inFIG. 28 .

FORMULATIONS, DOSES, AND ROUTES OF ADMINISTRATION

In carrying out a treatment method of the present disclosure, a synTaccan be formulated in a composition comprising a pharmaceuticallyacceptable excipient, and an immune checkpoint inhibitor can beformulated in a composition comprising a pharmaceutically acceptableexcipient. For simplicity, the term “active agent” is used below torefer to a synTac or an immune checkpoint inhibitor. In general, thesynTac and the immune checkpoint inhibitor are present in separatecompositions.

The composition may comprise a pharmaceutically acceptable excipient, avariety of which are known in the art and need not be discussed indetail herein. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example,“Remington: The Science and Practice of Pharmacy”, 19^(th) Ed. (1995),or latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy”, 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds 7^(th) ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3^(rd) ed. Amer. Pharmaceutical Assoc.

A pharmaceutical composition can comprise a synTac or an immunecheckpoint inhibitor, and a pharmaceutically acceptable excipient. Insome cases, a pharmaceutical composition will be suitable foradministration to a subject, e.g., will be sterile. For example, in somecases, a pharmaceutical composition will be suitable for administrationto a human subject, e.g., where the composition is sterile and is freeof detectable pyrogens and/or other toxins.

The compositions may comprise other components, such as pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate,and the like. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate, hydrochloride, sulfatesalts, solvates (e.g., mixed ionic salts, water, organics), hydrates(e.g., water), and the like.

For example, compositions may include aqueous solution, powder form,granules, tablets, pills, suppositories, capsules, suspensions, sprays,and the like. The composition may be formulated according to the variousroutes of administration described below.

Where an active agent (a synTac or an immune checkpoint inhibitor) isadministered as an injectable (e.g. subcutaneously, intraperitoneally,intramuscularly, and/or intravenously) directly into a tissue, aformulation can be provided as a ready-to-use dosage form, or asnon-aqueous form (e.g. a reconstitutable storage-stable powder) oraqueous form, such as liquid composed of pharmaceutically acceptablecarriers and excipients. The formulations may also be provided so as toenhance serum half-life of an active agent following administration. Forexample, the protein may be provided in a liposome formulation, preparedas a colloid, or other conventional techniques for extending serumhalf-life. A variety of methods are available for preparing liposomes,as described in, e.g., Szoka et al. 1980 Ann. Rev. Biophys. Bioeng.9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. Thepreparations may also be provided in controlled release or slow-releaseforms.

Other examples of formulations suitable for parenteral administrationinclude isotonic sterile injection solutions, anti-oxidants,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. For example, asubject pharmaceutical composition can be present in a container, e.g.,a sterile container, such as a syringe. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets.

The concentration of an active agent (a synTac or an immune checkpointinhibitor) in a formulation can vary widely (e.g., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight) and will usually be selected primarily based on fluidvolumes, viscosities, and patient-based factors in accordance with theparticular mode of administration selected and the patient's needs.

The present disclosure provides a container comprising an active agent(a synTac or an immune checkpoint inhibitor), e.g., a containercomprising a liquid composition comprising an active agent. Thecontainer can be, e.g., a syringe, an ampoule, and the like. In somecases, the container is sterile. In some cases, both the container andthe composition are sterile.

The present disclosure provides compositions, including pharmaceuticalcompositions, comprising an active agent (a synTac or an immunecheckpoint inhibitor). A composition can comprise: a) an active agent (asynTac or an immune checkpoint inhibitor); and b) an excipient, asdescribed above. In some cases, the excipient is a pharmaceuticallyacceptable excipient.

Compositions Comprising a Nucleic Acid or a Recombinant ExpressionVector

In some cases, a synTac is administered as a multimeric polypeptide perse. In other instances, one or more nucleic acids comprising nucleotidesequences encoding a synTac are administered, instead of administering asynTac as a multimeric polypeptide per se. The nucleic acid(s) can bepresent in a pharmaceutical composition. A pharmaceutical compositioncan comprise one or more recombinant expression vectors comprising theone or more nucleic acids. A wide variety of pharmaceutically acceptableexcipients is known in the art and need not be discussed in detailherein. Pharmaceutically acceptable excipients have been amply describedin a variety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy”, 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds 7^(th) ed., Lippincott,Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^(rd) ed. Amer. Pharmaceutical Assoc.

A pharmaceutical formulation can include a nucleic acid or recombinantexpression vector of the present disclosure in an amount of from about0.001% to about 90% (w/w). In the description of formulations, below,“nucleic acid or recombinant expression vector” will be understood toinclude a nucleic acid or recombinant expression vector comprisingnucleotide sequences encoding a synTac.

A nucleic acid or recombinant expression vector can be admixed,encapsulated, conjugated or otherwise associated with other compounds ormixtures of compounds; such compounds can include, e.g., liposomes orreceptor-targeted molecules. A nucleic acid or recombinant expressionvector can be combined in a formulation with one or more components thatassist in uptake, distribution and/or absorption.

A nucleic acid or recombinant expression vector composition can beformulated into any of many possible dosage forms such as, but notlimited to, tablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. A nucleic acid or recombinant expressionvector composition can also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers.

A formulation comprising a nucleic acid or recombinant expression vectorcan be a liposomal formulation. As used herein, the term “liposome”means a vesicle composed of amphiphilic lipids arranged in a sphericalbilayer or bilayers. Liposomes are unilamellar or multilamellar vesicleswhich have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes that can interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH sensitive or negatively charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomes canbe used to deliver a subject nucleic acid or recombinant expressionvector.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein by reference in its entirety.

The formulations and compositions of the present disclosure may alsoinclude surfactants. The use of surfactants in drug products,formulations and in emulsions is well known in the art. Surfactants andtheir uses are further described in U.S. Pat. No. 6,287,860.

In one embodiment, various penetration enhancers are included, to effectthe efficient delivery of nucleic acids. In addition to aiding thediffusion of non-lipophilic drugs across cell membranes, penetrationenhancers also enhance the permeability of lipophilic drugs. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants. Penetration enhancers andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tablets,or minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Suitable oral formulationsinclude those in which a subject antisense nucleic acid is administeredin conjunction with one or more penetration enhancers surfactants andchelators. Suitable surfactants include, but are not limited to, fattyacids and/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts and fatty acids and their uses are furtherdescribed in U.S. Pat. No. 6,287,860. Also suitable are combinations ofpenetration enhancers, for example, fatty acids/salts in combinationwith bile acids/salts. An exemplary suitable combination is the sodiumsalt of lauric acid, capric acid, and UDCA. Further penetrationenhancers include, but are not limited to, polyoxyethylene-9-laurylether, and polyoxyethylene-20-cetyl ether. Suitable penetrationenhancers also include propylene glycol, dimethylsulfoxide,triethanoiamine, N,N-dimethylacetamide, N,N-dimethylformamide,2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol, andAZONE™.

Treatment Methods

The present disclosure provides a treatment method, comprisingadministering a synTac and an immune checkpoint inhibitor. In somecases, the method comprises administering to an individual in needthereof: a) a first composition comprising a synTac; and b) a secondcomposition comprising an immune checkpoint inhibitor. In some cases, atreatment method of the present disclosure comprises administering to anindividual in need thereof: a) a first composition comprising one ormore recombinant expression vectors comprising nucleotide sequencesencoding a synTac; and b) a second composition comprising an immunecheckpoint inhibitor. In some cases, a treatment method of the presentdisclosure comprises administering to an individual in need thereof: a)a first composition comprising one or more mRNA molecules comprisingnucleotide sequences encoding a multimeric polypeptide; and b) a secondcomposition comprising an immune checkpoint inhibitor. In some cases,the immune checkpoint inhibitor is an antibody specific for an immunecheckpoint polypeptide.

Thus, for example, a treatment method of the present disclosure cancomprise co-administration of a synTac (e.g., a 4-1BBL synTac, IL-2synTac, etc., as described above) and an antibody specific for an immunecheckpoint. By “co-administration” is meant that both a synTac (e.g., a4-1BBL synTac, IL-2 synTac, etc., as described above) and an antibodyspecific for an immune checkpoint are administered to an individual,although not necessarily at the same time, in order to achieve atherapeutic effect that is the result of having administered both thesynTac and the immune checkpoint inhibitor. The administration of thesynTac (e.g., a 4-1BBL synTac, IL-2 synTac, etc.) and the antibodyspecific for an immune checkpoint can be substantially simultaneous,e.g., the synTac (e.g., a 4-1BBL synTac, IL-2 synTac, etc.) can beadministered to an individual within about 1 minute to about 24 hours(e.g., within about 1 minute, within about 5 minutes, within about 15minutes, within about 30 minutes, within about 1 hour, within about 4hours, within about 8 hours, within about 12 hours, or within about 24hours) of administration of the antibody specific for an immunecheckpoint. In some cases, a synTac (e.g., a 4-1BBL synTac, IL-2 synTac,etc.) is administered to an individual who is undergoing treatment withan antibody specific for an immune checkpoint. The administration of thesynTac (e.g., a 4-1BBL synTac, IL-2 synTac, etc.) and the antibodyspecific for an immune checkpoint can occur at different times and/or atdifferent frequencies.

Thus, for example, a treatment method of the present disclosure cancomprise co-administration of a 4-1BBL synTac and an antibody specificfor an immune checkpoint. By “co-administration” is meant that both a4-1BBL synTac and an antibody specific for an immune checkpoint areadministered to an individual, although not necessarily at the sametime, in order to achieve a therapeutic effect that is the result ofhaving administered both the synTac and the immune checkpoint inhibitor.The administration of the 4-1BBL synTac and the antibody specific for animmune checkpoint can be substantially simultaneous, e.g., the 4-1BBLsynTac can be administered to an individual within about 1 minute toabout 24 hours (e.g., within about 1 minute, within about 5 minutes,within about 15 minutes, within about 30 minutes, within about 1 hour,within about 4 hours, within about 8 hours, within about 12 hours, orwithin about 24 hours) of administration of the antibody specific for animmune checkpoint. In some cases, a 4-1BBL synTac is administered to anindividual who is undergoing treatment with an antibody specific for animmune checkpoint. The administration of the 4-1BBL synTac and theantibody specific for an immune checkpoint can occur at different timesand/or at different frequencies.

As another example, a treatment method of the present disclosure cancomprise co-administration of a synTac (e.g., an IL-2 synTac, asdescribed above) and an antibody specific for an immune checkpoint. By“co-administration” is meant that both an IL-2 synTac and an antibodyspecific for an immune checkpoint are administered to an individual,although not necessarily at the same time, in order to achieve atherapeutic effect that is the result of having administered both thesynTac and the immune checkpoint inhibitor. The administration of theIL-2 synTac and the antibody specific for an immune checkpoint can besubstantially simultaneous, e.g., the IL-2 synTac can be administered toan individual within about 1 minute to about 24 hours (e.g., withinabout 1 minute, within about 5 minutes, within about 15 minutes, withinabout 30 minutes, within about 1 hour, within about 4 hours, withinabout 8 hours, within about 12 hours, or within about 24 hours) ofadministration of the antibody specific for an immune checkpoint. Insome cases, an IL-2 synTac is administered to an individual who isundergoing treatment with an antibody specific for an immune checkpoint.The administration of the IL-2 synTac and the antibody specific for animmune checkpoint can occur at different times and/or at differentfrequencies.

The present disclosure provides a treatment method, comprisingadministering a synTac and an immune checkpoint inhibitor. A treatmentmethod of the present disclosure can modulate an activity of a target Tcell. In some cases, e.g., where the target T cell is a CD8 T cell, themultimeric polypeptide comprises Class I MHC polypeptides (e.g.,02-microglobulin and Class I MHC heavy chain). In some cases, e.g.,where the target T cell is a CD4⁺ T cell, the multimeric polypeptidecomprises Class II MHC polypeptides (e.g., Class II MHC α chain; ClassII MHC β chain).

Where a multimeric polypeptide includes an immunomodulatory polypeptidethat is an activating polypeptide, a method of the present disclosureactivates the epitope-specific T cell. In some instances, theepitope-specific T cell is a T cell that is specific for an epitopepresent on a cancer cell, and contacting the epitope-specific T cellwith the multimeric polypeptide increases cytotoxic activity of the Tcell toward the cancer cell. In some instances, the epitope-specific Tcell is a T cell that is specific for an epitope present on a cancercell, and a method of the present disclosure increases the number of theepitope-specific T cells.

In some instances, the epitope-specific T cell is a T cell that isspecific for an epitope present on a virus-infected cell, and a methodof the present disclosure increases cytotoxic activity of the T celltoward the virus-infected cell. In some instances, the epitope-specificT cell is a T cell that is specific for an epitope present on avirus-infected cell, and a method of the present disclosure increasesthe number of the epitope-specific T cells.

Where a multimeric polypeptide of the present disclosure includes animmunomodulatory polypeptide that is an inhibiting polypeptide, a methodof the present disclosure inhibits the epitope-specific T cell. In someinstances, the epitope-specific T cell is a self-reactive T cell that isspecific for an epitope present in a self antigen, and a method of thepresent disclosure reduces the number of the self-reactive T cells.

In some cases, the immunomodulatory polypeptide is an activatingpolypeptide, and the multimeric polypeptide activates theepitope-specific T cell. In some cases, the epitope is acancer-associated epitope, and the multimeric polypeptide increases theactivity of a T cell specific for the cancer-associate epitope.

In some cases, a treatment method of the present disclosure treats acancer in an individual having the cancerl. Thus, the present disclosureprovides a method of treating cancer in an individual, the methodcomprising administering to the individual: a) a multimeric polypeptideof the present disclosure, or one or more nucleic acids (e.g.,expression vectors; mRNA; etc.) comprising nucleotide sequences encodingthe multimeric polypeptide, where the multimeric polypeptide comprises aT-cell epitope that is a cancer epitope, and where the multimericpolypeptide comprises a stimulatory immunomodulatory polypeptide; and b)an immune checkpoint inhibitor. In some cases, “effective amounts” of amultimeric polypeptide and an immune checkpoint inhibitor are amountsthat, when administered in one or more doses to an individual in needthereof, reduce the number of cancer cells in the individual. Forexample, in some cases, “effective amounts” of a multimeric polypeptideand an immune checkpoint inhibitor are amounts that, when administeredin one or more doses to an individual in need thereof, reduce the numberof cancer cells in the individual by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%,compared to the number of cancer cells in the individual beforeadministration of the multimeric polypeptide and the immune checkpointinhibitor, or in the absence of administration with the multimericpolypeptide and the immune checkpoint inhibitor. In some cases,“effective amounts” of a multimeric polypeptide and an immune checkpointinhibitor are amounts that, when administered in one or more doses to anindividual in need thereof, reduce the number of cancer cells in theindividual to undetectable levels. In some cases, “effective amounts” ofa multimeric polypeptide and an immune checkpoint inhibitor are amountsthat, when administered in one or more doses to an individual in needthereof, reduce the tumor mass in the individual. For example, in somecases, “effective amounts” of a multimeric polypeptide and an immunecheckpoint inhibitor are amounts that, when administered in one or moredoses to an individual in need thereof, reduce the tumor mass in theindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%, compared to the tumor mass inthe individual before administration of the multimeric polypeptide andthe immune checkpoint inhibitor, or in the absence of administrationwith the multimeric polypeptide and the immune checkpoint inhibitor. Insome cases, “effective amounts” of a multimeric polypeptide and animmune checkpoint inhibitor are amounts that, when administered in oneor more doses to an individual in need thereof, increase survival timeof the individual. For example, in some cases, “effective amounts” of amultimeric polypeptide and an immune checkpoint inhibitor are amountsthat, when administered in one or more doses to an individual in needthereof, increase survival time of the individual by at least 1 month,at least 2 months, at least 3 months, from 3 months to 6 months, from 6months to 1 year, from 1 year to 2 years, from 2 years to 5 years, from5 years to 10 years, or more than 10 years, compared to the expectedsurvival time of the individual in the absence of administration withthe multimeric polypeptide and the immune checkpoint inhibitor.

In some instances, the epitope-specific T cell is a T cell that isspecific for an epitope present on a virus-infected cell, and a methodof the present disclosure increases cytotoxic activity of the T celltoward the virus-infected cell. In some instances, the epitope-specificT cell is a T cell that is specific for an epitope present on avirus-infected cell, and a method of the present disclosure increasesthe number of the epitope-specific T cells.

As noted above, in some cases, in carrying out a subject treatmentmethod, a multimeric polypeptide is administered to an individual inneed thereof, as the polypeptide per se. In other instances, in carryingout a subject treatment method, one or more nucleic acids comprisingnucleotide sequences encoding a multimeric polypeptide is/areadministering to an individual in need thereof. Thus, in otherinstances, one or more nucleic acids encoding a synTac is/areadministered to an individual in need thereof.

Dosages—synTac

A suitable dosage of a synTac can be determined by an attendingphysician or other qualified medical personnel, based on variousclinical factors. As is well known in the medical arts, dosages for anyone patient depend upon many factors, including the patient's size, bodysurface area, age, the particular polypeptide or nucleic acid to beadministered, sex of the patient, time, and route of administration,general health, and other drugs being administered concurrently. Amultimeric polypeptide (synTac) may be administered in amounts between 1ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5 mg/kg bodyweight to 5 mg/kg body weight; however, doses below or above thisexemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, it canalso be in the range of 1 g to 10 mg per kilogram of body weight perminute. A multimeric polypeptide can be administered in an amount offrom about 1 mg/kg body weight to 50 mg/kg body weight, e.g., from about1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kgbody weight to about 10 mg/kg body weight, from about 10 mg/kg bodyweight to about 15 mg/kg body weight, from about 15 mg/kg body weight toabout 20 mg/kg body weight, from about 20 mg/kg body weight to about 25mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kgbody weight, from about 30 mg/kg body weight to about 35 mg/kg bodyweight, from about 35 mg/kg body weight to about 40 mg/kg body weight,or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of a multimeric polypeptide is from 0.01μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of bodyweight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg perkg of body weight, from 100 μg to 10 mg per kg of body weight, or from100 μg to 1 mg per kg of body weight. Persons of ordinary skill in theart can easily estimate repetition rates for dosing based on measuredresidence times and concentrations of the administered agent in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein a multimeric polypeptide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, from 0.1 μg to 10 g per kg of body weight, from 1 g to 1g per kg of body weight, from 10 μg to 100 mg per kg of body weight,from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg perkg of body weight.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific multimeric polypeptide, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In some embodiments, multiple doses of a multimeric polypeptide (or anucleic acid or a recombinant expression vector encoding same) areadministered. The frequency of administration of a multimericpolypeptide can vary depending on any of a variety of factors, e.g.,severity of the symptoms, etc. For example, in some embodiments, amultimeric polypeptide is administered once per month, twice per month,three times per month, every other week (qow), once per week (qw), twiceper week (biw), three times per week (tiw), four times per week, fivetimes per week, six times per week, every other day (qod), daily (qd),twice a day (qid), or three times a day (tid).

The duration of administration of a multimeric polypeptide, e.g., theperiod of time over which a multimeric polypeptide is administered, canvary, depending on any of a variety of factors, e.g., patient response,etc. For example, a multimeric polypeptide can be administered over aperiod of time ranging from about one day to about one week, from abouttwo weeks to about four weeks, from about one month to about two months,from about two months to about four months, from about four months toabout six months, from about six months to about eight months, fromabout eight months to about 1 year, from about 1 year to about 2 years,or from about 2 years to about 4 years, or more.

A suitable dosage of a synTac can be determined by an attendingphysician or other qualified medical personnel, based on variousclinical factors. As is well known in the medical arts, dosages for anyone patient depend upon many factors, including the patient's size, bodysurface area, age, the particular polypeptide or nucleic acid to beadministered, sex of the patient, time, and route of administration,general health, and other drugs being administered concurrently. Amultimeric polypeptide (synTac) may be administered in amounts between 1ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5 mg/kg bodyweight to 5 mg/kg body weight; however, doses below or above thisexemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, it canalso be in the range of 1 g to 10 mg per kilogram of body weight perminute. A multimeric polypeptide can be administered in an amount offrom about 1 mg/kg body weight to 50 mg/kg body weight, e.g., from about1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kgbody weight to about 10 mg/kg body weight, from about 10 mg/kg bodyweight to about 15 mg/kg body weight, from about 15 mg/kg body weight toabout 20 mg/kg body weight, from about 20 mg/kg body weight to about 25mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kgbody weight, from about 30 mg/kg body weight to about 35 mg/kg bodyweight, from about 35 mg/kg body weight to about 40 mg/kg body weight,or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of a multimeric polypeptide is from 0.01μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of bodyweight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg perkg of body weight, from 100 μg to 10 mg per kg of body weight, or from100 μg to 1 mg per kg of body weight. Persons of ordinary skill in theart can easily estimate repetition rates for dosing based on measuredresidence times and concentrations of the administered agent in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein a multimeric polypeptide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, from 0.1 μg to 10 g per kg of body weight, from 1 g to 1g per kg of body weight, from 10 μg to 100 mg per kg of body weight,from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg perkg of body weight.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific multimeric polypeptide, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In some embodiments, multiple doses of a multimeric polypeptide (or anucleic acid or a recombinant expression vector encoding same) areadministered. The frequency of administration of a multimericpolypeptide can vary depending on any of a variety of factors, e.g.,severity of the symptoms, etc. For example, in some embodiments, amultimeric polypeptide is administered once per month, twice per month,three times per month, every other week (qow), once per week (qw), twiceper week (biw), three times per week (tiw), four times per week, fivetimes per week, six times per week, every other day (qod), daily (qd),twice a day (qid), or three times a day (tid).

The duration of administration of a multimeric polypeptide, e.g., theperiod of time over which a multimeric polypeptide is administered, canvary, depending on any of a variety of factors, e.g., patient response,etc. For example, a multimeric polypeptide can be administered over aperiod of time ranging from about one day to about one week, from abouttwo weeks to about four weeks, from about one month to about two months,from about two months to about four months, from about four months toabout six months, from about six months to about eight months, fromabout eight months to about 1 year, from about 1 year to about 2 years,or from about 2 years to about 4 years, or more.

Dosages—Immune Checkpoint Inhibitor

A suitable dosage of an immune checkpoint inhibitor can be determined byan attending physician or other qualified medical personnel, based onvarious clinical factors. As is well known in the medical arts, dosagesfor any one patient depend upon many factors, including the patient'ssize, body surface area, age, the particular polypeptide or nucleic acidto be administered, sex of the patient, time, and route ofadministration, general health, and other drugs being administeredconcurrently. An immune checkpoint inhibitor may be administered inamounts between 1 ng/kg body weight and 20 mg/kg body weight per dose,e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, it canalso be in the range of 1 μg to 10 mg per kilogram of body weight perminute. An immune checkpoint inhibitor can be administered in an amountof from about 1 mg/kg body weight to 50 mg/kg body weight, e.g., fromabout 1 mg/kg body weight to about 5 mg/kg body weight, from about 5mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kgbody weight to about 15 mg/kg body weight, from about 15 mg/kg bodyweight to about 20 mg/kg body weight, from about 20 mg/kg body weight toabout 25 mg/kg body weight, from about 25 mg/kg body weight to about 30mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kgbody weight, from about 35 mg/kg body weight to about 40 mg/kg bodyweight, or from about 40 mg/kg body weight to about 50 mg/kg bodyweight.

In some cases, a suitable dose of an immune checkpoint inhibitor is from0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg ofbody weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, orfrom 100 μg to 1 mg per kg of body weight. Persons of ordinary skill inthe art can easily estimate repetition rates for dosing based onmeasured residence times and concentrations of the administered agent inbodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein an immune checkpoint inhibitoris administered in maintenance doses, ranging from 0.01 μg to 100 g perkg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μgto 1 g per kg of body weight, from 10 μg to 100 mg per kg of bodyweight, from 100 g to 10 mg per kg of body weight, or from 100 μg to 1mg per kg of body weight.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific immune checkpoint inhibitor, the severity ofthe symptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In some embodiments, multiple doses of an immune checkpoint inhibitorare administered. The frequency of administration an immune checkpointinhibitor can vary depending on any of a variety of factors, e.g.,severity of the symptoms, etc. For example, in some embodiments, amultimeric polypeptide is administered once per month, twice per month,three times per month, every other week (qow), once per week (qw), twiceper week (biw), three times per week (tiw), four times per week, fivetimes per week, six times per week, every other day (qod), daily (qd),twice a day (qid), or three times a day (tid).

The duration of administration of an immune checkpoint inhibitor, e.g.,the period of time over which an immune checkpoint inhibitor isadministered, can vary, depending on any of a variety of factors, e.g.,patient response, etc. For example, an immune checkpoint inhibitor canbe administered over a period of time ranging from about one day toabout one week, from about two weeks to about four weeks, from about onemonth to about two months, from about two months to about four months,from about four months to about six months, from about six months toabout eight months, from about eight months to about 1 year, from about1 year to about 2 years, or from about 2 years to about 4 years, ormore.

The following are non-limiting examples.

Pembrolizumab can be administered to an individual in need thereof in anamount of 2 mg/kg every 3 weeks. Pembrolizumab can be administered to anindividual in need thereof in an amount of 200 mg every 3 weeks. In somecases, a method of the present disclosure provides for a reduced amountof an anti-PD1 that needs to be administered to achieve clinicalbenefit. For example, in some cases, the amount of pembrolizumab thatneeds to be administered to achieve clinical benefit can be reduced byfrom 10% to 50%, or more than 50%, compared to the amount ofpembrolizumab that needs to be administered to achieve clinical benefitin the absence of treatment with a synTac.

Nivolumab can be administered to an individual in need thereof in anamount of 3 mg/kg every 2 weeks. Nivolumab can be administered to anindividual in need thereof in an amount of 240 mg every 2 weeks. In somecases, a method of the present disclosure provides for a reduced amountof an anti-PD1 that needs to be administered to achieve clinicalbenefit. For example, in some cases, the amount of nivolumab that needsto be administered to achieve clinical benefit can be reduced by from10% to 50%, or more than 50%, compared to the amount of nivolumab thatneeds to be administered to achieve clinical benefit in the absence oftreatment with a synTac.

Atezolizumab can be administered to an individual in need thereof in anamount of 1200 mg every 3 weeks. In some cases, a method of the presentdisclosure provides for a reduced amount of an anti-PD1 that needs to beadministered to achieve clinical benefit. For example, in some cases,the amount of atezolizumab that needs to be administered to achieveclinical benefit can be reduced by from 10% to 50%, or more than 50%,compared to the amount of atezolizumab that needs to be administered toachieve clinical benefit in the absence of treatment with a synTac.

Ipilimumab can be administered to an individual in need thereof in anamount of 3 mg/kg every 3 weeks. Ipilimumab can be administered to anindividual in need thereof in an amount of 10 mg/kg every 3 weeks.Ipilimumab can be administered to an individual in need thereof in anamount of 10 mg/kg every 12 weeks. In some cases, a method of thepresent disclosure provides for a reduced amount of an anti-PD1 thatneeds to be administered to achieve clinical benefit. For example, insome cases, the amount of ipilimumab that needs to be administered toachieve clinical benefit can be reduced by from 10% to 50%, or more than50%, compared to the amount of ipilimumab that needs to be administeredto achieve clinical benefit in the absence of treatment with a synTac.

Routes of Administration

An active agent (a multimeric polypeptide; an immune checkpointinhibitor) is administered to an individual using any available methodand route suitable for drug delivery, including in vivo and ex vivomethods, as well as systemic and localized routes of administration. Insome cases, a synTac is administered by a first route of administration;and an immune checkpoint inhibitor is administered by a second route ofadministration that is different from the first route of administration.In some cases, a synTac and an immune checkpoint inhibitor areadministered by the same routes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intratumoral, peritumoral, intramuscular, intratracheal,intracranial, subcutaneous, intradermal, topical application,intravenous, intraarterial, rectal, nasal, oral, and other enteral andparenteral routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the multimericpolypeptide, the immune checkpoint inhibitor and/or the desired effect.

In some cases, a multimeric polypeptide is administered intravenously;and an immune checkpoint inhibitor intravenously. In some cases, amultimeric polypeptide is administered intramuscularly; and an immunecheckpoint inhibitor intramuscularly. In some cases, a multimericpolypeptide is administered locally; and an immune checkpoint inhibitorlocally. In some cases, a multimeric polypeptide is administeredintratumorally; and an immune checkpoint inhibitor intratumorally. Insome cases, a multimeric polypeptide is administered peritumorally; andan immune checkpoint inhibitor peritumorally. In some cases, amultimeric polypeptide is administered intracranially; and an immunecheckpoint inhibitor intracranially. In some cases, a multimericpolypeptide is administered subcutaneously; and an immune checkpointinhibitor subcutaneously.

In some cases, a multimeric polypeptide is administered intravenously;and an immune checkpoint inhibitor peritumorally. In some cases, amultimeric polypeptide is administered intramuscularly; and an immunecheckpoint inhibitor intravenously. In some cases, a multimericpolypeptide is administered systemically; and an immune checkpointinhibitor locally. In some cases, a multimeric polypeptide isadministered intratumorally; and an immune checkpoint inhibitorintravenously. In some cases, a multimeric polypeptide is administeredsystemically; and an immune checkpoint inhibitor peritumorally. In somecases, a multimeric polypeptide is administered intravenously; and animmune checkpoint inhibitor intracranially. In some cases, a multimericpolypeptide is administered subcutaneously; and an immune checkpointinhibitor intravenously.

A multimeric polypeptide and an immune checkpoint inhibitor can beadministered to a host using any available conventional methods androutes suitable for delivery of conventional drugs, including systemicor localized routes. In general, routes of administration contemplatedfor use in a method of the present disclosure include, but are notnecessarily limited to, enteral, parenteral, and inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intratumoral, peritumoral, and intravenous routes, i.e.,any route of administration other than through the alimentary canal.Parenteral administration can be carried to effect systemic or localdelivery of an active agent (a synTac or an immune checkpointinhibitor). Where systemic delivery is desired, administration caninvolve intravenous delivery.

Subjects Suitable for Treatment

Subjects suitable for treatment with a method of the present disclosureinclude individuals who have cancer, including individuals who have beendiagnosed as having cancer, individuals who have been treated for cancerbut who failed to respond to the treatment, and individuals who havebeen treated for cancer and who initially responded but subsequentlybecame refractory to the treatment. Subjects suitable for treatment witha method of the present disclosure include individuals who have aninfection (e.g., an infection with a pathogen such as a bacterium, avirus, a protozoan, etc.), including individuals who have been diagnosedas having an infection, and individuals who have been treated for aninfection but who failed to respond to the treatment. Subjects suitablefor treatment with a method of the present disclosure includeindividuals who have bacterial infection, including individuals who havebeen diagnosed as having a bacterial infection, and individuals who havebeen treated for a bacterial infection but who failed to respond to thetreatment. Subjects suitable for treatment with a method of the presentdisclosure include individuals who have a viral infection, includingindividuals who have been diagnosed as having a viral infection, andindividuals who have been treated for a viral infection but who failedto respond to the treatment. Subjects suitable for treatment with amethod of the present disclosure include individuals who have anautoimmune disease, including individuals who have been diagnosed ashaving an autoimmune disease, and individuals who have been treated fora autoimmune disease but who failed to respond to the treatment.

In some cases, a method of the present disclosure comprisesadministering a synTac to an individual who is undergoing treatment withan immune checkpoint inhibitor. In some cases, a method of the presentdisclosure comprises administering a synTac to an individual who isundergoing treatment with an anti-PD1 antibody. For example, in somecases, a method of the present disclosure comprises administering asynTac to an individual who is undergoing treatment with pembrolizumab.As another example, in some cases, a method of the present disclosurecomprises administering a synTac to an individual who is undergoingtreatment with nivolumab. In some cases, a method of the presentdisclosure comprises administering a synTac to an individual who isundergoing treatment with an anti-PD-L1 antibody. For example, in somecases, a method of the present disclosure comprises administering asynTac to an individual who is undergoing treatment with atezolizumab.In some cases, a method of the present disclosure comprisesadministering a synTac to an individual who is undergoing treatment withan anti-CTLA4 antibody. For example, in some cases, a method of thepresent disclosure comprises administering a synTac to an individual whois undergoing treatment with ipilimumab. As another example, in somecases, a method of the present disclosure comprises administering asynTac to an individual who is undergoing treatment with tremelimumab.

In some cases, e.g., where the epitope is an HPV epitope, a subjectsuitable for treatment with a method of the present disclosure is anindividual who has been diagnosed as having an HPV-associated cancer oran HPV-attributable cancer. HPV-associated and HPV-attributable cancersinclude, e.g., head and neck cancer; cervical cancer; and genitoanalcancer.

Methods of Selectively Delivering a Costimulatory Polypeptide Togetherwith an Immune Checkpoint Inhibitor to an Individual

The present disclosure thus provides a method of delivering acostimulatory polypeptide such as IL-2, or a reduced-affinity variant ofa naturally occurring costimulatory polypeptide such as an IL-2 variantdisclosed herein, to a selected T cell or a selected T cell population,e.g., in a manner such that a TCR specific for a given epitope istargeted, together with co-administration of a checkpoint inhibitor soas to provide the patient with the therapeutic effect of both theselective delivery of the costimulatory polypeptide and the checkpointinhibitor. The present disclosure provides a method of delivering acostimulatory polypeptide such as IL-2, or a reduced-affinity variant ofa naturally occurring costimulatory polypeptide such as an IL-2 variantdisclosed herein, selectively to a target T cell bearing a TCR specificfor the epitope present in a multimeric polypeptide of the presentdisclosure, together with co-administration of a checkpoint inhibitor soas to provide the patient with the therapeutic effect of both theselective delivery of the costimulatory polypeptide and the checkpointinhibitor. The method comprises contacting a population of T cells witha multimeric polypeptide of the present disclosure. The population of Tcells can be a mixed population that comprises: i) the target T cell;and ii) non-target T cells that are not specific for the epitope (e.g.,T cells that are specific for an epitope(s) other than the epitope towhich the epitope-specific T cell binds). The epitope-specific T cell isspecific for the epitope-presenting peptide present in the multimericpolypeptide, and binds to the peptide HLA complex or peptide MHC complexprovided by the multimeric polypeptide. Contacting the population of Tcells with the multimeric polypeptide delivers the costimulatorypolypeptide (e.g., IL-2 or a reduced-affinity variant of IL-2) presentin the multimeric polypeptide selectively to the T cell(s) that arespecific for the epitope present in the multimeric polypeptide. Thecheckpoint inhibitor is co-administered with the multimeric polypeptide(either together or at different times before and/or afteradministration of the multimeric polypeptide) so as to provide thepatient with the therapeutic effect of both the selective delivery ofthe costimulatory polypeptide and the checkpoint inhibitor.

Thus, the present disclosure provides a method of delivering to apatient (i) a checkpoint inhibitor as described above and (ii) acostimulatory polypeptide such as IL-2, or a reduced-affinity variant ofa naturally occurring costimulatory polypeptide such as an IL-2 variantdisclosed herein, or a combination of both, selectively to a target Tcell, the method comprising contacting a mixed population of T cellswith a multimeric polypeptide of the present disclosure. The mixedpopulation of T cells comprises the target T cell and non-target Tcells. The target T cell is specific for the epitope present within themultimeric polypeptide. Contacting the mixed population of T cells witha multimeric polypeptide of the present disclosure delivers thecostimulatory polypeptide(s) present within the multimeric polypeptideto the target T cell. The co-administration of the checkpoint inhibitorwith the multimeric polypeptide (either together or at different timesbefore and/or after administration of the multimeric polypeptide) thusprovides the patient with the therapeutic effect of both the selectivedelivery of the costimulatory polypeptide and the checkpoint inhibitor.

For example, a multimeric polypeptide of the present disclosure iscontacted with a population of T cells comprising: i) a target T cell(s)that is specific for the epitope present in the multimeric polypeptide;and ii) a non-target T cell(s), e.g., a T cell(s) that is specific for asecond epitope(s) that is not the epitope present in the multimericpolypeptide. Contacting the population results in selective delivery ofthe costimulatory polypeptide(s) (e.g., naturally-occurringcostimulatory polypeptide (e.g., naturally occurring IL-2) orreduced-affinity variant of a naturally occurring costimulatorypolypeptide (e.g., an IL-2 variant disclosed herein)), which is presentin the multimeric polypeptide, to the target T cell. Thus, e.g., lessthan 50%, less than 40%, less than 30%, less than 25%, less than 20%,less than 15%, less than 10%, less than 5%, or less than 4%, 3%, 2% or1%, of the non-target T cells bind the multimeric polypeptide and, as aresult, the costimulatory polypeptide (e.g., IL-2 or IL-2 variant)present in the multimeric polypeptides is substantially not delivered tothe non-target T cells. The co-administration of the checkpointinhibitor with the multimeric polypeptide (either together or atdifferent times before and/or after administration of the multimericpolypeptide) thus provides the patient with the therapeutic effect ofboth the selective delivery of the costimulatory polypeptide and thecheckpoint inhibitor.

In some cases, the population of T cells is in vitro. In some cases, thepopulation of T cells is in vitro, and a biological response (e.g., Tcell activation and/or expansion and/or phenotypic differentiation) ofthe target T cell population to the multimeric polypeptide of thepresent disclosure is elicited in the context of an in vitro culture.For example, a mixed population of T cells can be obtained from anindividual, and can be contacted with the multimeric polypeptide invitro. Such contacting can comprise single or multiple exposures of thepopulation of T cells to a defined dose(s) and/or exposure schedule(s).In some cases, said contacting results in selectively binding/activatingand/or expanding target T cells within the population of T cells, andresults in generation of a population of activated and/or expandedtarget T cells. As an example, a mixed population of T cells can beperipheral blood mononuclear cells (PBMC). For example, PBMC from apatient can be obtained by standard blood drawing and PBMC enrichmenttechniques before being exposed to 0.1-1000 nM of a multimericpolypeptide of the present disclosure under standard lymphocyte cultureconditions. At time points before, during, and after exposure of themixed T cell population at a defined dose and schedule, the abundance oftarget T cells in the in vitro culture can be monitored by specificpeptide-MHC multimers and/or phenotypic markers and/or functionalactivity (e.g. cytokine ELISpot assays). In some cases, upon achievingan optimal abundance and/or phenotype of antigen specific cells invitro, all or a portion of the population of activated and/or expandedtarget T cells is administered to the individual (the individual fromwhom the mixed population of T cells was obtained), which individualalready has received administration of a checkpoint inhibitor prior tothe administration of the target T cells and/or will receiveadministration of a checkpoint inhibitor after the administration oftarget T cells.

In some cases, the population of T cells is in vitro. For example, amixed population of T cells is obtained from an individual, and iscontacted with a multimeric polypeptide of the present disclosure invitro. Such contacting, which can comprise single or multiple exposuresof the T cells to a defined dose(s) and/or exposure schedule(s) in thecontext of in vitro cell culture, can be used to determine whether themixed population of T cells includes T cells that are specific for theepitope presented by the multimeric polypeptide. The presence of T cellsthat are specific for the epitope of the multimeric polypeptide can bedetermined by assaying a sample comprising a mixed population of Tcells, which population of T cells comprises T cells that are notspecific for the epitope (non-target T cells) and may comprise T cellsthat are specific for the epitope (target T cells). Known assays can beused to detect activation and/or proliferation of the target T cells,thereby providing an ex vivo assay that can determine whether aparticular multimeric polypeptide (synTac) possesses an epitope thatbinds to T cells present in the individual and thus whether themultimeric polypeptide has potential use as a therapeutic compositionfor that individual. Suitable known assays for detection of activationand/or proliferation of target T cells include, e.g., flow cytometriccharacterization of T cell phenotype and/or antigen specificity and/orproliferation. Such an assay to detect the presence of epitope-specificT cells, e.g., a companion diagnostic, can further include additionalassays (e.g. effector cytokine ELISpot assays) and/or appropriatecontrols (e.g. antigen-specific and antigen-nonspecific multimericpeptide-HLA staining reagents) to determine whether the multimericpolypeptide is selectively binding/activating and/or expanding thetarget T cell. Thus, for example, the present disclosure provides amethod of detecting, in a mixed population of T cells obtained from anindividual, the presence of a target T cell that binds an epitope ofinterest, the method comprising: a) contacting in vitro the mixedpopulation of T cells with a multimeric polypeptide of the presentdisclosure, wherein the multimeric polypeptide comprises the epitope ofinterest; and b) detecting activation and/or proliferation of T cells inresponse to said contacting, wherein activated and/or proliferated Tcells indicates the presence of the target T cell. Alternatively, and/orin addition, if activation and/or expansion (proliferation) of thedesired T cell population is obtained using the multimeric polypeptide,then all or a portion of the population of T cells comprising theactivated/expanded T cells can be administered back to the individual(who has received administration of a checkpoint inhibitor beforeadministration of the T cells and/or will receive administration of acheckpoint inhibitor after administration of the T cells) as a therapy.

In some instances, the population of T cells is in vivo in anindividual. In such instances, a method of the present disclosure forselectively delivering a costimulatory polypeptide (e.g., IL-2 or areduced-affinity IL-2) to an epitope-specific T cell comprisesadministering the multimeric polypeptide to the individual.

The epitope-specific T cell to which a costimulatory polypeptide (e.g.,IL-2 or a reduced-affinity IL-2) is being selectively delivered is alsoreferred to herein as a “target T cell.” In some cases, the target Tcell is a regulatory T cell (Treg). In some cases, the Treg inhibits orsuppresses activity of an autoreactive T cell.

In some cases, the target T cell is a cytotoxic T cell. For example, thetarget T cell can be a cytotoxic T cell specific for a cancer epitope(e.g., an epitope presented by a cancer cell).

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure designated Aspects 1-122,Aspects A-Z, Aspects AA-ZZ, and Aspects AAA-BBB, are provided below. Aswill be apparent to those of skill in the art upon reading thisdisclosure, each of the individually numbered aspects may be used orcombined with any of the preceding or following individually numberedaspects. This is intended to provide support for all such combinationsof aspects and is not limited to combinations of aspects explicitlyprovided below:

Aspect 1. A variant IL-2 polypeptide comprising an amino acid sequencehaving at least 85% amino acid sequence identity to set forth in SEQ IDNO:1, wherein the variant IL-2 polypeptide has one or more amino acidsubstitutions relative to set forth in SEQ ID NO:1, and wherein thevariant IL-2 polypeptide exhibits reduced binding affinity to an IL-2receptor (IL2R) comprising alpha, beta, and gamma polypeptides havingamino acid sequences depicted in FIG. 3A-3C, compared to the bindingaffinity of the IL-2 amino acid sequence set forth in one of SEQ ID NO:1for the IL2R.

Aspect 2. The variant IL2 polypeptide of aspect 1, wherein the variantcomprises a substitution of one or more of E15, H16, D20, F42, Y45, andQ126.

Aspect 3. The variant IL2 polypeptide of aspect 1 or aspect 2, whereinthe variant immunomodulatory polypeptide exhibits from less than 10% toless than 50% of the binding affinity exhibited by the IL2 amino acidsequence set forth in SEQ ID NO:1 for the IL2R.

Aspect 4. The variant IL2 polypeptide of any one of aspects 1-3, whereinthe variant comprises substitutions of F42 with Ala, Gly, Val, Ile, orLeu.

Aspect 5. The variant IL2 polypeptide of any one of aspects 1-3, whereinthe variant comprises substitutions of F42 and D20.

Aspect 6. The variant IL2 polypeptide of any one of aspects 1-3, whereinthe variant comprises substitutions of F42 and H16.

Aspect 7. The variant IL2 polypeptide of any one of aspects 1-3, whereinthe variant comprises substitutions of F42, D20, and Y45; or wherein thevariant comprises substitutions of F42, H16, and Q126.

Aspect 8. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first major histocompatibility complex (MHC)            polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a            non-Ig scaffold,    -   wherein the multimeric polypeptide comprises one or more        immunomodulatory domains, wherein the one or more        immunomodulatory domain is:        -   A) at the C-terminus of the first polypeptide;        -   B) at the N-terminus of the second polypeptide;        -   C) at the C-terminus of the second polypeptide; or        -   D) at the C-terminus of the first polypeptide and at the            N-terminus of the second polypeptide,    -   wherein at least one of the one or more immunomodulatory domains        is a variant IL2 polypeptide of any one of aspects 1-7, and    -   wherein the multimeric polypeptide exhibits reduced binding        affinity to an IL-2 receptor (IL2R) comprising alpha, beta, and        gamma polypeptides having amino acid sequences depicted in FIG.        3A-3C, compared to the binding affinity of a control multimeric        polypeptide comprising the IL2 amino acid sequence set forth in        SEQ ID NO:1 for the IL2R polypeptide.

Aspect 9. The multimeric polypeptide of aspect 8, wherein:

-   -   a) the first polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the epitope;        -   ii) the first MHC polypeptide; and        -   iii) the variant IL2 polypeptide; and    -   b) the second polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the second MHC polypeptide; and        -   ii) the Ig Fc polypeptide.

Aspect 10. The multimeric polypeptide of aspect 8, wherein:

-   -   a) the first polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the epitope; and        -   ii) the first MHC polypeptide; and    -   b) the second polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the variant IL2 polypeptide;        -   ii) the second MHC polypeptide; and        -   iii) the Ig Fc polypeptide.

Aspect 11. The multimeric polypeptide of aspect 8, wherein:

-   -   a) the first polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the epitope; and        -   ii) the first MHC polypeptide; and    -   b) the second polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the second MHC polypeptide; and        -   ii) the variant IL2 polypeptide.

Aspect 12. The multimeric polypeptide of aspect 8, wherein:

-   -   a) the first polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the epitope; and        -   ii) the first MHC polypeptide; and    -   b) second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) the variant IL2 polypeptide; and        -   ii) the second MHC polypeptide.

Aspect 13. The multimeric polypeptide of aspect 8, wherein:

-   -   a) the first polypeptide comprises, in order from N-terminus to        C-terminus:        -   i) the epitope;        -   ii) the first MHC polypeptide; and        -   iii) the variant IL2 polypeptide; and    -   b) the second polypeptide comprises the second MHC polypeptide.

Aspect 14. The multimeric polypeptide of aspect 8, wherein the non-Igscaffold is an XTEN polypeptide, a transferrin polypeptide, anelastin-like polypeptide, a silk-like polypeptide, or asilk-elastin-like polypeptide.

Aspect 15. The multimeric polypeptide of any one of aspects 8-14,wherein the first MHC polypeptide is a 02-microglobulin polypeptide; andwherein the second MHC polypeptide is an MHC class I heavy chainpolypeptide.

Aspect 16. The multimeric polypeptide of aspect 15, wherein the02-microglobulin polypeptide comprises an amino acid sequence having atleast 85% amino acid sequence identity to one of the amino acidsequences set forth in FIG. 6 .

Aspect 17. The multimeric polypeptide of aspect 15, wherein the MHCclass I heavy chain polypeptide is an HLA-A, an HLA-B, or an HLA-C heavychain.

Aspect 18. The multimeric polypeptide of aspect 15, wherein the MHCclass I heavy chain polypeptide comprises an amino acid sequence havingat least 85% amino acid sequence identity to the amino acid sequence setforth in one of FIG. 5A-5C.

Aspect 19. The multimeric polypeptide of any one of aspects 8-14,wherein the first MHC polypeptide is an MHC Class II alpha chainpolypeptide; and wherein the second MHC polypeptide is an MHC class IIbeta chain polypeptide.

Aspect 20. The multimeric polypeptide of any one of aspects 8-19,wherein the epitope is a T-cell epitope.

Aspect 21. The multimeric polypeptide of any one of aspects 8-13 and15-20, wherein multimeric polypeptide comprises an Fc polypeptide, andwherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fcpolypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fcpolypeptide, or an IgM Fc polypeptide.

Aspect 22. The multimeric polypeptide of aspect 21, wherein the Ig Fcpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to an amino acid sequence depicted in FIG. 4A-4C.

Aspect 23. The multimeric polypeptide of any one of aspects 8-22,wherein the first polypeptide and the second polypeptide arenon-covalently associated.

Aspect 24. The multimeric polypeptide of any one of aspects 8-22,wherein the first polypeptide and the second polypeptide are covalentlylinked to one another.

Aspect 25. The multimeric polypeptide of aspect 24, wherein the covalentlinkage is via a disulfide bond.

Aspect 26. The multimeric polypeptide of aspect 25, wherein the firstMHC polypeptide or a linker between the epitope and the first MHCpolypeptide comprises an amino acid substitution to provide a first Cysresidue, and the second MHC polypeptide comprises an amino acidsubstitution to provide a second Cys residue, and wherein the disulfidelinkage is between the first and the second Cys residues.

Aspect 27. The multimeric polypeptide of any one of aspects 8-26,comprising a linker interposed between the epitope and the first MHCpolypeptide.

Aspect 28. The multimeric polypeptide of any one of aspects 8-26,comprising a linker interposed between the MHC polypeptide and theimmunomodulatory polypeptide.

Aspect 29. The multimeric polypeptide of any one of aspects 8-28,comprising 2 variant IL2 polypeptides.

Aspect 30. The multimeric polypeptide of any one of aspects 8-28,comprising 3 variant IL2 polypeptides.

Aspect 31. The multimeric polypeptide of aspect 29 or aspect 30, whereinthe 2 or 3 variant IL2 polypeptides are in tandem, and wherein themultimeric polypeptide comprises a linker between the variant IL2polypeptides.

Aspect 32. The multimeric polypeptide of any one of aspects 8-28,wherein the variant IL2 comprises a substitution of one or more of E15,H16, D20, F42, Y45, and Q126/

Aspect 33. The multimeric polypeptide of any one of aspects 8-28,wherein the variant IL2 comprises a substitution of F42 with Ala, Gly,Val, Ile, or Leu.

Aspect 34. The multimeric polypeptide of aspect 33, wherein the variantIL2 comprises substitutions of F42 and D20.

Aspect 35. The multimeric polypeptide of aspect 33, wherein the variantIL2 comprises substitutions of F42 and H16.

Aspect 36. The multimeric polypeptide of aspect 33, wherein the variantIL2 comprises substitutions of F42, D20, and Y45; or wherein the variantIL-2 comprising substitutions of F42, H16, and Q126.

Aspect 37. A nucleic acid comprising a nucleotide sequence encoding arecombinant polypeptide,

-   -   i) wherein the recombinant polypeptide comprises, in order from        N-terminus to C-terminus:    -   a) an epitope;    -   b) a first major histocompatibility complex (MHC) polypeptide;    -   c) an immunomodulatory polypeptide;    -   d) a proteolytically cleavable linker or a ribosome skipping        signal;    -   e) a second MHC polypeptide; and    -   f) an immunoglobulin (Ig) Fc polypeptide;    -   wherein the immunomodulatory polypeptide is a variant        immunomodulatory polypeptide of any one of aspects 1-7; or    -   ii) wherein the recombinant polypeptide comprises, in order from        N-terminus to C-terminus:    -   a) an epitope;    -   b) a first MHC polypeptide;    -   c) a proteolytically cleavable linker or a ribosome skipping        signal;    -   d) an immunomodulatory polypeptide    -   e) a second MHC polypeptide; and    -   f) an Ig Fc polypeptide,    -   wherein the immunomodulatory polypeptide is a variant        immunomodulatory polypeptide of any one of aspects 1-7.

Aspect 38. The nucleic acid of aspect 37, wherein the first MHCpolypeptide is a 02-microglobulin polypeptide; and wherein the secondMHC polypeptide is an MHC class I heavy chain polypeptide.

Aspect 39. The nucleic acid of aspect 38, wherein the 02-microglobulinpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to one of the amino acid sequences set forth inFIG. 6 .

Aspect 40. The nucleic acid of aspect 38, wherein the MHC class I heavychain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.

Aspect 41. The nucleic acid of aspect 40, wherein the MHC class I heavychain polypeptide comprises an amino acid sequence having at least 85%amino acid sequence identity to the amino acid sequence set forth in anyone of FIG. 5A-5C.

Aspect 42. The nucleic acid of aspect 37, wherein the first MHCpolypeptide is an MHC Class II alpha chain polypeptide; and wherein thesecond MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect 43. The nucleic acid of any one of aspects 37-43, wherein theepitope is a T-cell epitope.

Aspect 44. The nucleic acid of any one of aspects 37-43, wherein the IgFc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, anIgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, oran IgM Fc polypeptide.

Aspect 45. The nucleic acid of aspect 44, wherein the Ig Fc polypeptidecomprises an amino acid sequence having at least 85% amino acid sequenceidentity to an amino acid sequence depicted in FIGS. 4A-4C.

Aspect 46. The nucleic acid of any one of aspects 37-45, wherein thevariant IL2 immunomodulatory polypeptide comprises a substitution of oneor more of E15, H16, D20, F42, Y45, and Q126.

Aspect 47. The nucleic acid of any one of aspects 37-46, wherein themultimeric polypeptide comprises a second immunomodulatory polypeptideselected from a CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and HVEM.

Aspect 48. The nucleic acid of any one of aspects 37-47, wherein theproteolytically cleavable linker or ribosome skipping signal comprisesan amino acid sequence selected from:

-   -   a) LEVLFQGP (SEQ ID NO:88);    -   b) ENLYTQS (SEQ ID NO:90);    -   c) a furin cleavage site;    -   d) LVPR (SEQ ID NO:89);    -   e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:91);    -   f) GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:92);    -   g) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:93); and    -   h) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:94).

Aspect 49. The nucleic acid of aspect 31, wherein the recombinantpolypeptide comprises, in order from N-terminus to C-terminus:

-   -   a) a first leader peptide;    -   b) the epitope;    -   c) the first MHC polypeptide;    -   d) the immunomodulatory polypeptide;    -   e) the proteolytically cleavable linker or ribosome skipping        signal;    -   f) a second leader peptide;    -   g) the second MHC polypeptide; and    -   h) the immunoglobulin (Ig) Fc polypeptide.

Aspect 50. The nucleic acid of aspect 49, wherein the first leaderpeptide and the second leader peptide is a β2-M leader peptide.

Aspect 51. The nucleic acid of any one of aspects 37-50, wherein thenucleotide sequence is operably linked to a transcriptional controlelement.

Aspect 52. The nucleic acid of aspect 51, wherein the transcriptionalcontrol element is a promoter that is functional in a eukaryotic cell.

Aspect 53. The nucleic acid of any one of aspects 37-52, wherein thefirst MHC polypeptide or a linker between the epitope and the first MHCpolypeptide comprises an amino acid substitution to provide a first Cysresidue, and the second MHC polypeptide comprises an amino acidsubstitution to provide a second Cys residue, and wherein the first andthe second Cys residues provide for a disulfide linkage between thefirst MHC polypeptide and the second MHC polypeptide.

Aspect 54. A recombinant expression vector comprising the nucleic acidof any one of aspects 37-52.

Aspect 55. The recombinant expression vector of aspect 54, wherein thevector is a viral vector or a non-viral vector.

Aspect 56. A host cell genetically modified with the recombinantexpression vector of aspect 48-55.

Aspect 57. The host cell of aspect 56, wherein the host cell is invitro.

Aspect 58. The host cell of aspect 57, wherein the host cell isgenetically modified such that the cell does not produce an endogenousMHC β2-microglobulin polypeptide.

Aspect 59. A composition comprising:

-   -   a) a first nucleic acid comprising a nucleotide sequence        encoding a first polypeptide comprising, in order from        N-terminus to C-terminus:        -   i) an epitope;        -   ii) a first MHC polypeptide; and        -   iii) an immunomodulatory domain,    -   wherein the immunomodulatory domain is a variant IL2 polypeptide        of any one of aspects 1-7; and    -   b) a first nucleic acid comprising a nucleotide sequence        encoding a second polypeptide comprising, in order from        N-terminus to C-terminus:        -   i) a second MHC polypeptide; and        -   ii) an Ig Fc polypeptide.

Aspect 60. A composition comprising:

-   -   a) a first nucleic acid comprising a nucleotide sequence        encoding a first polypeptide comprising, in order from        N-terminus to C-terminus:        -   i) an epitope; and        -   ii) a first MHC polypeptide; and    -   b) a first nucleic acid comprising a nucleotide sequence        encoding a second polypeptide comprising, in order from        N-terminus to C-terminus:        -   i) an immunomodulatory domain, wherein the immunomodulatory            domain is a variant IL2 polypeptide of any one of aspects            1-7;        -   ii) a second MHC polypeptide; and        -   iii) an Ig Fc polypeptide.

Aspect 61. The composition of aspect 59 or aspect 60, wherein the firstand/or the second nucleic acid is present in a recombinant expressionvector.

Aspect 62. A host cell genetically modified with the composition of anyone of aspects 59-61.

Aspect 63. A method of producing the multimeric polypeptide of any oneof aspects 8-36, the method comprising:

-   -   a) culturing the host cell of any one of aspects 56-58 and 62 in        vitro in a culture medium under conditions such that the host        cell synthesizes the multimeric polypeptide; and    -   b) isolating the multimeric polypeptide from the host cell        and/or from the culture medium.

Aspect 64. The method of aspect 63, wherein the second polypeptidecomprises an affinity tag, and wherein said isolating comprisescontacting the multimeric polypeptide produced by the cell with abinding partner for the affinity tag, wherein the binding partner isimmobilized, thereby immobilizing the multimeric polypeptide.

Aspect 65. The method of aspect 64, comprising eluting the immobilizedmultimeric polypeptide.

Aspect 66. A method of selectively activating an epitope-specific Tcell, the method comprising contacting the T cell with the multimericpolypeptide of any one of aspects 8-36, wherein said contactingselectively activates the epitope-specific T cell.

Aspect 67. The method of aspect 66, wherein said contacting is in vitro.

Aspect 68. The method of aspect 66, wherein said contacting is in vivo.

Aspect 69. The method of aspect 66, wherein the epitope is acancer-associated epitope, and wherein said administering selectivelyincreases the activity of a T cell specific for the cancer-associateepitope.

Aspect 70. A method of treating cancer in an individual, the methodcomprising administering to the individual an effective amount of:

-   -   a) the multimeric polypeptide of any one of aspects 8-36; or    -   b) one or more recombinant expression vectors comprising        nucleotide sequences encoding the multimeric polypeptide of any        one of aspects 8-36; or    -   c) one or more mRNAs comprising nucleotide sequences encoding        the multimeric polypeptide of any one of aspects 8-36,    -   wherein the epitope is a cancer-associated epitope, and wherein        said administering effective to selectively activate a cancer        epitope-specific T cell in an individual.

Aspect 71. The method of aspect 70, wherein said administering issubcutaneous.

Aspect 72. The method of aspect 70, wherein said administering isintravenous.

Aspect 73. The method of aspect 70, wherein said administering isperitumoral.

Aspect 74. The method of aspect 70, wherein said administering issystemic.

Aspect 75. The method of aspect 70, wherein said administering is distalto a treatment site.

Aspect 76. The method of aspect 70, wherein said administering is local.

Aspect 77. The method of aspect 70, wherein said administering is at ornear a treatment site.

Aspect 78. A composition comprising:

-   -   a) the multimeric polypeptide of any one of aspects 8-36; and    -   b) a pharmaceutically acceptable excipient.

Aspect 79. A composition comprising:

-   -   a) the nucleic acid of any one of aspects 37-53 or the        recombinant expression vector of aspect 54 or 55; and    -   b) a pharmaceutically acceptable excipient.

Aspect 80. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a 02-microglobulin (β2M) polypeptide comprising the            amino acid sequence depicted in FIG. 34A; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a variant IL-2 polypeptide of any one of aspects 1-7;        -   ii) a major histocompatibility comples (MHC) heavy chain            polypeptide comprising the amino acid sequence depicted in            FIG. 34C; and        -   iii) an IgG1 Fc polypeptide comprising one or more amino            acid substitutions selected from N297A, L234A, L235A, L234F,            L235E, and P331S (N77A, L14A, L15A, L14F, L15E, and P111S,            respectively, based on the amino acid numbering depicted in            FIG. 33A).

Aspect 81. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an N297A substitution (N77A based on the aminoacid numbering depicted in FIG. 33A).

Aspect 82. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an L234A substitution and an L235A substitution(L14A and L15A based on the amino acid numbering depicted in FIG. 33A).

Aspect 83. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an L234F substitution and an L235E substitution(L14F and L15E based on the amino acid numbering depicted in FIG. 33A).

Aspect 84. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an L234F substitution, an L235E substitution, anda P331S (L14F, L15E, and P111S substitutions based on the amino acidnumbering depicted in FIG. 33A).

Aspect 85. The multimeric polypeptide of any one of aspects 80-84,wherein the second polypeptide comprises two copies of the variant IL-2polypeptide.

Aspect 86. The multimeric polypeptide of any one of aspects 80-85,wherein the first polypeptide comprises a peptide linker between theepitope and the β2M polypeptide.

Aspect 87. The multimeric polypeptide of any one of aspects 80-86,wherein the second polypeptide comprises a peptide linker between one ormore of:

-   -   a) a first copy of the variant IL-2 polypeptide and a second        copy of the variant IL-2 polypeptide;    -   b) the variant IL-2 polypeptide and the MHC heavy chain        polypeptide; and    -   c) between the MHC heavy chain polypeptide and the IgG1 Fc        polypeptide.

Aspect 88. The multimeric polypeptide of aspect 86 or aspect 87, whereinthe peptide linker is selected from (GGGGS)₃, (GGGGS)₄, and AAAGG.

Aspect 89. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a β2-microglobulin polypeptide comprising the amino acid            sequence depicted in FIG. 34A; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a variant IL-2 polypeptide comprising the amino acid            sequence depicted in FIG. 34B;        -   ii) a major histocompatibility comples (MHC) heavy chain            polypeptide comprising the amino acid sequence depicted in            FIG. 34C; and        -   iii) an IgG1 Fc polypeptide comprising one or more amino            acid substitutions selected from N297A, L234A, L235A, L234F,            L235E, and P331S (N77A, L14A, L15A, L14F, L15E, and P111S,            respectively, based on the amino acid numbering depicted in            FIG. 33A).

Aspect 90. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an N297A substitution (N77A based on the aminoacid numbering depicted in FIG. 33A).

Aspect 91. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an L234A substitution and an L235A substitution(L14A and L15A based on the amino acid numbering depicted in FIG. 33A).

Aspect 92. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an L234F substitution and an L235E substitution(L14F and L15E based on the amino acid numbering depicted in FIG. 33A).

Aspect 93. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an L234F substitution, an L235E substitution, anda P331S substitution (L14F, L15E, and P111S based on the amino acidnumbering depicted in FIG. 33A).

Aspect 94. The multimeric polypeptide of any one of aspects 89-93,wherein the second polypeptide comprises two copies of the variant IL-2polypeptide.

Aspect 95. The multimeric polypeptide of any one of aspects 89-94,wherein the first polypeptide comprises a peptide linker between theepitope and the β2M polypeptide.

Aspect 96. The multimeric polypeptide of any one of aspects 89-95,wherein the second polypeptide comprises a peptide linker between one ormore of:

-   -   a) a first copy of the variant IL-2 polypeptide and a second        copy of the variant IL-2 polypeptide;    -   b) the variant IL-2 polypeptide and the MHC heavy chain        polypeptide; and    -   c) between the MHC heavy chain polypeptide and the IgG1 Fc        polypeptide.

Aspect 97. The multimeric polypeptide of aspect 95 or aspect 96, whereinthe peptide linker is selected from (GGGGS)₃, (GGGGS)₄, and AAAGG.

Aspect 98. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope comprising the amino acid sequence YMLDLQPETT            (SEQ ID NO:77);        -   ii) a 02-microglobulin polypeptide comprising the amino acid            sequence depicted in FIG. 34A; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a variant IL-2 polypeptide comprising the amino acid            sequence depicted in FIG. 34B;        -   ii) a major histocompatibility comples (MHC) heavy chain            polypeptide comprising the amino acid sequence depicted in            FIG. 34C; and        -   iii) an IgG1 Fc polypeptide comprising the amino acid            sequence depicted in FIG. 33A, 33B, 33C, or 33D.

Aspect 99. The multimeric polypeptide of aspect 98, wherein the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33B.

Aspect 100. The multimeric polypeptide of aspect 98, wherein the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33C.

Aspect 101. The multimeric polypeptide of aspect 98, wherein the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33D.

Aspect 102. The multimeric polypeptide of any one of aspects 98-101,wherein the second polypeptide comprises two copies of the variant IL-2polypeptide.

Aspect 103. The multimeric polypeptide of any one of aspects 98-102,wherein the first polypeptide comprises a peptide linker between theepitope and the β2M polypeptide.

Aspect 104. The multimeric polypeptide of any one of aspects 98-103,wherein the second polypeptide comprises a peptide linker between one ormore of:

-   -   a) a first copy of the variant IL-2 polypeptide and a second        copy of the variant IL-2 polypeptide;    -   b) the variant IL-2 polypeptide and the MHC heavy chain        polypeptide; and    -   c) between the MHC heavy chain polypeptide and the IgG1 Fc        polypeptide.

Aspect 105. The multimeric polypeptide of aspect 103 or aspect 104,wherein the peptide linker is selected from (GGGGS)₃, (GGGGS)₄, andAAAGG.

Aspect 106. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising the amino acid sequence        depicted in FIG. 31 ;    -   b) a second polypeptide comprising the amino acid sequence        depicted in FIG. 22 .

Aspect 107. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising the amino acid sequence        depicted in FIG. 31 ;    -   b) a second polypeptide comprising the amino acid sequence        depicted in FIG. 25 .

Aspect 108. A multimeric polypeptide comprising:

-   -   a) a first polypeptide comprising the amino acid sequence        depicted in FIG. 31 ;    -   b) a second polypeptide comprising the amino acid sequence        depicted in FIG. 28 .

Aspect 109. A pharmaceutical composition comprising:

-   -   a) a multimeric polypeptide according to any one of aspects        80-108; and    -   b) a pharmaceutically acceptable excipient.

Aspect 110. One or more nucleic acids comprising nucleotide sequencesencoding the first and/or the second polypeptide of the multimericpolypeptide according to any one of aspects 80-108.

Aspect 111. The one or more nucleic acids of aspect 110, wherein thenucleic acid(s) is/are present in recombinant expression vectors.

Aspect 112. A method of selectively activating an epitope-specific Tcell, the method comprising contacting the T cell with the multimericpolypeptide of any one of aspects 80-108, wherein said contactingselectively activates the epitope-specific T cell.

Aspect 113. The method of aspect 112, wherein said contacting is invitro.

Aspect 114. The method of aspect 112, wherein said contacting is invivo.

Aspect 115. A method comprising administering to an individual aneffective amount of:

-   -   a) the multimeric polypeptide of any one of aspects 80-108; or    -   b) one or more recombinant expression vectors comprising        nucleotide sequences encoding the multimeric polypeptide of any        one of aspects 80-108; or    -   c) one or more mRNAs comprising nucleotide sequences encoding        the multimeric polypeptide of any one of aspects 80-108, wherein        said administering induces a T cell response to epitope in the        individual.

Aspect 116. The method of aspect 115, wherein said administering issubcutaneous.

Aspect 117. The method of aspect 115, wherein said administering isintravenous.

Aspect 118. The method of aspect 115, wherein said administering issystemic.

Aspect 119. The method of aspect 115, wherein said administering isintramuscular.

Aspect 120. The method of aspect 115, wherein said administering isdistal to a treatment site.

Aspect 121. The method of aspect 115, wherein said administering islocal.

Aspect 122. The method of aspect 115, wherein said administering is ator near a treatment site.

Aspect A. A method of modulating an immune response an individual inneed thereof, the method comprising administering to the individual amultimeric polypeptide and an immune checkpoint inhibitor,

-   -   wherein the multimeric polypeptide comprises:    -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first major histocompatibility complex (MHC)            polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a            non-Ig scaffold,    -   wherein the multimeric polypeptide comprises one or more        immunomodulatory polypeptides, wherein the one or more        immunomodulatory polypeptides is:        -   A) at the C-terminus of the first polypeptide;        -   B) at the N-terminus of the second polypeptide;        -   C) at the C-terminus of the second polypeptide; or        -   D) at the C-terminus of the first polypeptide and at the            N-terminus of the second polypeptide; and    -   wherein said administering modulates the immune response in the        individual.

Aspect B. A treatment method comprising administering to an individual amultimeric polypeptide and an immune checkpoint inhibitor,

-   -   wherein the multimeric polypeptide comprises:    -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first major histocompatibility complex (MHC)            polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a            non-Ig scaffold,    -   wherein the multimeric polypeptide comprises one or more        immunomodulatory polypeptides, wherein the one or more        immunomodulatory polypeptides is:        -   A) at the C-terminus of the first polypeptide;        -   B) at the N-terminus of the second polypeptide;        -   C) at the C-terminus of the second polypeptide; or        -   D) at the C-terminus of the first polypeptide and at the            N-terminus of the second polypeptide; and    -   wherein said administering treats the individual.

Aspect C. A method of treating cancer in an individual, the methodcomprising administering to the individual a multimeric polypeptide andan immune checkpoint inhibitor,

-   -   wherein the multimeric polypeptide comprises:    -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first major histocompatibility complex (MHC)            polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a            non-Ig scaffold,    -   wherein the multimeric polypeptide comprises one or more        immunomodulatory polypeptides, wherein the one or more        immunomodulatory polypeptides is:        -   A) at the C-terminus of the first polypeptide;        -   B) at the N-terminus of the second polypeptide;        -   C) at the C-terminus of the second polypeptide; or        -   D) at the C-terminus of the first polypeptide and at the            N-terminus of the second polypeptide; and    -   wherein said administering treats the cancer in the individual.

Aspect D. A treatment method comprising administering to an individual amultimeric polypeptide, where the individual is undergoing treatmentwith an immune checkpoint inhibitor,

-   -   wherein the multimeric polypeptide comprises:    -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first major histocompatibility complex (MHC)            polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a            non-Ig scaffold,    -   wherein the multimeric polypeptide comprises one or more        immunomodulatory polypeptides, wherein the one or more        immunomodulatory polypeptides is:        -   A) at the C-terminus of the first polypeptide;        -   B) at the N-terminus of the second polypeptide;        -   C) at the C-terminus of the second polypeptide; or        -   D) at the C-terminus of the first polypeptide and at the            N-terminus of the second polypeptide; and    -   wherein said administering treats the individual.

Aspect E. The method of any one of Aspects A-D, wherein the multimericpolypeptide comprises:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first MHC polypeptide; and        -   iii) an immunomodulatory domain; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) an Ig Fc polypeptide.

Aspect F. The method of any one of Aspects A-D, wherein the multimericpolypeptide comprises:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope; and        -   ii) a first MHC polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an immunomodulatory domain;        -   iii) a second MHC polypeptide; and        -   ii) an immunoglobulin (Ig) Fc polypeptide.

Aspect G. The method of any one of Aspects A-D, wherein the multimericpolypeptide comprises:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope; and        -   ii) a first MHC polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) an Ig Fc polypeptide; and        -   iii) an immunomodulatory domain.

Aspect H. The method of any one of Aspects A-D, wherein the multimericpolypeptide comprises:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope; and        -   ii) a first MHC polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide; and        -   ii) an immunomodulatory domain.

Aspect I. The method of any one of Aspects A-D, wherein the multimericpolypeptide comprises:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope; and        -   ii) a first MHC polypeptide; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an immunomodulatory domain; and        -   ii) a second MHC polypeptide.

Aspect J. The method of any one of Aspects A-D, wherein the multimericpolypeptide comprises:

-   -   a) a first polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) an epitope;        -   ii) a first MHC polypeptide; and        -   iii) an immunomodulatory domain; and    -   b) a second polypeptide comprising, in order from N-terminus to        C-terminus:        -   i) a second MHC polypeptide.

Aspect K. The method of any one of Aspects A-D, wherein the non-Igscaffold of the multimeric polypeptide is an XTEN polypeptide, atransferrin polypeptide, an Fc receptor polypeptide, an elastin-likepolypeptide, a silk-like polypeptide, or a silk-elastin-likepolypeptide.

Aspect L. The method of any one of aspects A-K, wherein the first MHCpolypeptide of the multimeric polypeptide is a 02-microglobulinpolypeptide; and wherein the second MHC polypeptide is an MHC class Iheavy chain polypeptide.

Aspect M. The method of aspect L, wherein the 02-microglobulinpolypeptide of the multimeric polypeptide comprises an amino acidsequence having at least 85% amino acid sequence identity to one of theamino acid sequences set forth in FIG. 6 .

Aspect N. The method of aspect L, wherein the MHC class I heavy chainpolypeptide of the multimeric polypeptide is an HLA-A, an HLA-B, or anHLA-C heavy chain.

Aspect O. The method of aspect 11, wherein the MHC class I heavy chainpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to the amino acid sequence set forth in one ofFIG. 5A-5C.

Aspect P. The method of any one of aspects A-K, wherein the first MHCpolypeptide of the multimeric polypeptide is an MHC Class II alpha chainpolypeptide; and wherein the second MHC polypeptide is an MHC class IIbeta chain polypeptide.

Aspect Q. The method of any one of aspects A-P, wherein the epitope is aT-cell epitope.

Aspect R. The method of any one of aspects A-J, wherein multimericpolypeptide of the multimeric polypeptide comprises an Fc polypeptide,and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fcpolypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fcpolypeptide, or an IgM Fc polypeptide.

Aspect S. The method of aspect R, wherein the Ig Fc polypeptidecomprises an amino acid sequence having at least 85% amino acid sequenceidentity to an amino acid sequence depicted in FIG. 4A-4C.

Aspect T. The method of any one of aspects A-S, wherein the firstpolypeptide and the second polypeptide of the multimeric polypeptide arenon-covalently associated.

Aspect U. The method of any one of aspects A-S, wherein the firstpolypeptide and the second polypeptide of the multimeric polypeptide arecovalently linked.

Aspect V. The method of aspect U, wherein the covalent linkage is via adisulfide bond.

Aspect W. The method of aspect V, wherein the first MHC polypeptide or alinker between the epitope and the first MHC polypeptide of themultimeric polypeptide comprises an amino acid substitution to provide afirst Cys residue, and the second MHC polypeptide of the multimericpolypeptide comprises an amino acid substitution to provide a second Cysresidue, and wherein the disulfide linkage is between the first and thesecond Cys residues.

Aspect X. The method of any one of aspects A-K, wherein the multimericpolypeptide comprises a linker between the epitope and the first MHCpolypeptide, between the immunomodulatory polypeptide and the MHCpolypeptide, or between the MHC polypeptide and the Ig Fc.

Aspect Y. The method of any one of aspects A-K, wherein theimmunomodulatory polypeptide of the multimeric polypeptide is selectedfrom a 4-1BBL polypeptide, a B7-1 polypeptide; a B7-2 polypeptide, anICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2polypeptide.

Aspect Z. The method of any one of aspects A-Y, wherein the multimericpolypeptide comprises 2 or more immunomodulatory polypeptides.

Aspect AA. The method of aspect Z, wherein the 2 or moreimmunomodulatory polypeptides are in tandem.

Aspect BB. The method of any one of aspects A-Z and AA, wherein theimmunomodulatory polypeptide is selected from a 4-1BBL polypeptide, aCD80 polypeptide, a CD86 polypeptide, an IL-2 polypeptide, a B7-1polypeptide; a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40Lpolypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasLpolypeptide, and a PD-L2 polypeptide.

Aspect CC. The method of any one of aspects A-Z and AA-BB, wherein theimmunomodulatory polypeptide is a variant immunomodulatory polypeptidehas one or more amino acid substitutions relative to the naturallyoccurring form of the immunomodulatory polypeptide, and wherein thevariant immunomodulatory polypeptide exhibits reduced binding affinityto a co-modulatory polypeptide to which the naturally occurring form ofthe immunomodulatory polypeptide binds.

Aspect DD. The method of any one of aspects A-Z and AA-BB, wherein theimmunomodulatory polypeptide is a variant IL-2 polypeptide comprising anamino acid sequence having at least 85% amino acid sequence identity toset forth in SEQ ID NO:1, wherein the variant IL-2 polypeptide has oneor more amino acid substitutions relative to set forth in SEQ ID NO:1,and wherein the variant IL-2 polypeptide exhibits reduced bindingaffinity to an IL-2 receptor (IL2R) comprising alpha, beta, and gammapolypeptides having amino acid sequences depicted in FIG. 3A-3C,compared to the binding affinity of the IL-2 amino acid sequence setforth in one of SEQ ID NO:1 for the IL2R.

Aspect EE. The method of aspect DD, wherein the variant IL-2 polypeptidecomprises a substitution of one or more of E15, H16, D20, F42, Y45, andQ126.

Aspect FF. The method of aspect EE, wherein the variant IL-2 polypeptidecomprises:

-   -   a) substitutions of F42 and D20;    -   b) substitutions of F42 and H16;    -   c) substitutions of F42, D20, and Y45; or    -   d) substitutions of F42, H16, and Q126.

Aspect GG. The method of any one of aspects A-Z and AA-FF, wherein themultimeric polypeptide comprises an Ig Fc polypeptide comprising one ormore amino acid substitutions selected from N297A, L234A, L235A, L234F,L235E, and P331S.

Aspect HH. The method of aspect GG, wherein the Ig Fc polypeptidecomprises:

-   -   a) an N297A substitution;    -   b) an L234A substitution and an L235A substitution;    -   c) an L234F substitution and an L235E substitution; or    -   d) an L234F substitution, an L235E substitution, and a P33IS        substitution.

Aspect II. The method of any one of aspects A-Z and AA-HH, wherein theepitope of the multimeric polypeptide comprises the amino acid sequenceYMLDLQPETT (SEQ ID NO:77).

Aspect JJ. The method of any one of aspects A-Z and AA-HH, wherein the02-microglobulin polypeptide of the multimeric polypeptide comprises theamino acid sequence depicted in FIG. 34A.

Aspect KK. The method of any one of aspects A-Z and AA-JJ, wherein themajor histocompatibility complex(MHC) heavy chain polypeptide of themultimeric polypeptide comprises the amino acid sequence depicted inFIG. 34C.

Aspect LL. The method of any one of aspects A-Z and AA-KK, wherein theimmune checkpoint inhibitor is an antibody specific for the immunecheckpoint inhibitor.

Aspect MM. The method of aspect LL, wherein the antibody is a monoclonalantibody.

Aspect NN. The method of aspect KK or aspect LL, wherein the antibodycomprises at least one humanized light chain and/or heavy chainframework region.

Aspect OO. The method of aspect LL, wherein the antibody comprises an Fcpolypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fcpolypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fcpolypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide.

Aspect PP. The method of aspect LL, wherein the antibody is an Fvfragment, a nanobody, or a Fab fragment.

Aspect QQ. The method of any one of aspects LL-PP, wherein the immunecheckpoint inhibitor is an antibody specific for an immune checkpointinhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40,GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (alsoknown as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3,VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.

Aspect RR. The method of any one of aspects LL-PP, wherein the immunecheckpoint inhibitor is an antibody specific for PD1.

Aspect SS. The method of aspect RR, wherein the antibody ispembrolizumab, nivolumab, pidilizumab, or BMS-39886.

Aspect TT. The method of any one of aspects LL-PP, wherein the immunecheckpoint inhibitor is an antibody specific for PD-L1.

Aspect UU. The method of aspect TT, wherein the antibody is durvalumab,atezolizumab, KN035, or avelumab.

Aspect VV. The method of any one of aspects LL-PP, wherein the immunecheckpoint inhibitor is an antibody specific for CTLA4.

Aspect WW. The method of aspect VV, wherein the antibody is ipilimumabor tremelimumab.

Aspect XX. The method of any one of aspects A-Z and AA-WW, wherein themultimeric polypeptide and the immune checkpoint inhibitor areadministered by the same route of administration.

Aspect YY. The method of any one of aspects A-Z and AA-WW, wherein themultimeric polypeptide and the immune checkpoint inhibitor areadministered by different routes of administration.

Aspect ZZ. The method of any one of aspects A-Z and AA-YY, wherein themultimeric polypeptide is administered by a route of administrationselected from subcutaneous, intravenous, peritumoral, and intramuscular.

Aspect AAA. The method of any one of aspects A-Z and AA-YY, wherein theimmune checkpoint inhibitor is administered by a route of administrationselected from subcutaneous, intravenous, peritumoral, and intramuscular.

Aspect BBB. The method of any one of aspects A-Z, AA-ZZ, and AAA,wherein the individual is a human.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); kiloDalton(s), kDa;i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

Example 1: Production of IL-2/synTac

Production of IL-2/synTac by transiently transfected mammalian cells wasanalyzed. As shown in FIG. 7A, production levels (in mg/L culturemedium) of two different IL-2/synTacs, 6-7 days following transienttransfection of the cells, was greater than 90 mg/L.

The IL-2/synTacs produced by the mammalian cells was purified, andsubjected to reducing and non-reducing polyacrylamide gelelectrophoresis. The results are depicted in FIG. 7B. Sizes are given inkDa.

IL-2/synTacs were generated, in which the IL-2 polypeptide was in the“light chain” (i.e., the polypeptide comprising MHC Class I light chain;e.g., β2M) or in the “heavy chain” (i.e., the polypeptide comprising MHCClass I heavy chain). Expression levels and stability of theIL-2/synTacs were analyzed.

The synTacs were produced in mammalian cells. As shown in FIG. 8A, theIL-2/synTac comprising IL-2 on the heavy chain was produced at levelsabout 25-fold higher than the level of the IL-2/synTac comprising IL-2on the light chain.

The IL-2/synTacs produced by mammalian cells were subjected to reducingand non-reducing polyacrylamide gel electrophoresis; and the gels werestained with Coomassie blue. As shown in FIG. 8B, the IL-2/synTaccomprising IL-2 on the heavy chain was more stable than the IL-2/synTaccomprising IL-2 on the light chain. Sizes are given in kDa.

Expression levels of IL-2/synTacs comprising variant IL-2 were assessed.FIG. 9 depicts the expression level of IL-2/syn-Tacs, in which the IL-2is wild-type (wt), or comprises various combinations of F42A, D20K,Q126A, E15A, Y45A, and H16A. The expression levels are expressed aspercent change relative to expression levels of a synTac with wild-typeIL-2.

The effect of the copy number of IL-2 in an IL-2/synTac on expressionlevels was evaluated. IL-2/synTacs comprising one copy (1×), two copies(2×) or three copies (3×) in the synTac. The various IL-2/synTacs wereproduced in mammalian cells, and expression levels were assayed. Thedata are depicted in FIG. 10 . IL-2/synTacs with one or two copies ofIL-2 exhibit similar expression levels, while an IL-2/synTac with threecopies of IL-2 exhibited lower expression levels. Expression levels areexpressed as fold change relative to the expression level of theIL-2/synTac with a single copy of IL-2.

Example 2: In Vitro Activity of IL-2/synTac

To achieve maximal specificity of targeting through a T-cell receptor,the affinity of the co-stimulatory polypeptide for its ligand should belower than the affinity of MHC for the TCR. The peptide/MHC affinity forTCR can be about 10 μM.

An IL-2/synTac was generated, comprising two copies of a variant IL-2comprising F42A and H16A substitutions. Costimulatory signaling inducedby the IL-2/synTac was tested on antigen-specific CD8⁺ T cells andnon-specific CD8⁺ T cells. Antigen-specific CD8⁺ T cells andnon-specific CD8⁺ T cells were contacted with various concentrations ofthe IL-2/synTac.

As shown in FIG. 11 , the IL-2/synTac induced costimulatory signaling inantigen-specific CD8⁺ T cells at a much lower concentration than innon-specific CD8⁺ T cells.

Selectivity of IL-2/synTac binding was tested. CD8⁺ T cells wereisolated from spleens of LCMV or OT1 mice. The CD8⁺ T cells wereincubated with IL-2/synTacs at various concentrations, and allowed tobind for 20 minutes. The IL-2/synTacs comprise IgG2a Fc. Binding ofIL-2/synTacs to the CD8⁺ T cells was detected using phycoerythrin(PE)-labeled anti-IgG2a antibody. PE fluorescence was detected usingflow cytometry to determine the percent of cells bound to IL-2/synTac.

As shown in FIG. 12 , IL-2/synTac binds in an antigen-specific manner toLCMV CD8⁺ T cells, but does not exhibit significant binding to OT1 CD8⁺T cells. Thus, IL-2/synTac selectively binds to CD8⁺ T cells specificfor the epitope present in the IL-2/synTac.

It was determined whether an IL-2/synTac selectively activates target Tcells. CD8⁺ T cells were isolated from spleens of LCMV or OT1 mice. TheIL-2/synTacs used included either the F42A single amino acidsubstitution, or the F42A and H16A substitutions. The CD8⁺ T cells werestimulated with IL-2/synTacs at various concentrations for 20 minutes.The cells were then stained with PE-labelled anti-phospho-STAT5antibody. PE fluorescence was detected using flow cytometry to determinethe percent of cells that are phospho-STAT5 positive, wherephospho-STAT5 is a marker of activation.

As shown in FIG. 13 , IL-2/synTac induced CD8⁺ stimulation (as indicatedby the % phospho-STAT5-positive cells) in antigen-specific (LCMV) CD8⁺ Tcells at much lower concentrations than in non-specific (BL6) CD8⁺ Tcells.

The specific activity of various IL-2/synTacs was analyzed. IL-2/synTacscomprising a single copy of IL-2, two copies of IL-2, or three copies ofIL-2, where the IL-2 comprised various combinations of F42A, D20K,Q126A, E15A, H16A, and Y45A substitutions, were tested at variousconcentrations for stimulation of CD8⁺ antigen-specific (LCMV) ornon-specific (BL6) cells. The percent phospho-signal transducer andactivator of transcription 5 (pSTAT5)-positive was determined. The dataare depicted in FIG. 14A-14F.

Example 3: In Vivo Activity of IL-2/synTac

The in vivo activity of IL-2/synTac was tested. The in vivo fold changein antigen-specific CD8⁺ T cells was tested, following administration ofphosphate buffered saline (PBS), recombinant IL-2 (rIL-2), or anIL-2/synTac of the present disclosure. The data are shown in FIG. 15 ,left panel. The data indicate that IL-2/synTac is 10 times more potentthan rIL-2.

The in vivo specificity of IL-2/synTac was tested. Antigen-specific andnon-antigen-specific responses following administration of PBS, rIL-2,or IL-2/synTac was assessed. The data are expressed as percent of lymphnode cells that were antigen-specific or antigen non-specific followingadministration of PBS, rIL-2, or IL-2/synTac. As depicted in FIG. 15 ,right panel, IL-2/synTac induced an antigen-specific response (expressedas % maximum dilution of carboxyfluorescein succinimidyl ester (CFSE),an index of T cell proliferation). In contrast, the response induced byrIL-2 was not antigen-specific.

A dose response assay was conducted. IL-2/synTac (F42A, H16A) wasadministered intraperitoneally at concentrations of 4 mg/kg, 8 mg/kg,and 16 mg/kg. The results are shown in FIG. 16A. As shown in FIG. 16A,IL-2/synTac administered at 4 mg/kg or 8 mg/kg gave similar results;IL-2/synTac administered at 16 mg/kg induced the most potentimmunostimulatory activity.

The effect of route of administration of IL-2/synTac was tested.IL-2/synTac (F42A, H16A) was administered at 4 mg/kg, eithersubcutaneously (SubQ) or intraperitoneally (IP). As shown in FIG. 16B,subcutaneous administration resulted in a more potent immunostimulatoryactivity than IP administration.

The effect of IL-2 copy number on efficacy was determined. IL-2/synTacscomprising a single copy of IL-2 (F42A, H16A) or two copies of IL-2(F42A, H16A) were injected into mice with tumors bearing an HPV E7epitope. The epitope included in the IL-2/synTacs was the HPV E7epitope. As shown in FIGS. 17A and 17B, an IL-2/synTac comprising twocopies of IL-2(F42A, H16A) were more effective at reducing tumor sizethan an IL-2/synTac comprising only a single copy of IL-2(F42A, H16A).

Example 4: PK/PD and Stability Studies of IL-2/synTac

Pharmacokinetic (PK) analysis of IL-2/synTac was carried out.IL-2/synTac (F42A, D20K, H16A) was administered IP at 10 mg/kg. Atvarious time points post-administration, serum samples were obtained andthe level of IL-2/synTac was measured in the serum samples. As shown inFIG. 18 , the serum half-life of the IL-2/synTac was about 4 hours.

IL-2/synTac was injected IP into a C57BL/6 mouse at 10 mg/kg, and serumwas collected two hours after injections. The IL-2/synTac included aHis₆ tag. 100 ng of the input protein, or the equivalent of 40 μl ofserum, was subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), and probed with an anti-(His)₆ antibody oran anti-β-2M antibody. The results, depicted in FIG. 19 , show thatIL-2/synTac remains stable and intact for at least 2 hours in vivo.

IL-2/synTac was kept at 4° C. or 37° C. for 5 days. 0.5 mg of eachsample (at 10 mg/ml) was analyzed by size exclusion chromatography. Asshown in FIG. 20 , IL-2/synTac is stable and intact for at least 5 daysat 4° C. or 37° C.

Example 5: Effect of an IL-2/synTac and an Anti-PD1 Antibody on TumorVolume

As shown in FIG. 35 , administration of an IL-2/synTac and an anti-PD1antibody to a mouse having a tumor reduced tumor volume.

Example 6: Generation and Characterization of synTac Polypeptides withVariant 4-1BBL

synTac polypeptides were synthesized and characaterized. The followingsynTac polypeptides were tested for activity on ovalbumin (OVA)-specificT cells:

-   -   1) Syn83/51. The light chain of Syn83/51 comprises: a) an OVA        T-cell epitope; b) amino acids 50-254 of a wild-type 4-1BBL        polypeptide; and c) β2M; and the heavy chain of Syn83/51        comprises: a) MHC heavy chain; and b) Ig Fc.    -   2) Syn239/345. The light chain of Syn239/345 comprises: a) an        OVA T-cell epitope; b) a trimer of amino acids 80-254 of        wild-type 4-1BBL; and c) β2M; and the heavy chain of Syn239/345        comprises: a) MHC heavy chain; and b) IgG2a Fc.    -   3) Syn341/348. The light chain of Syn341/348 comprises: a) an        OVA T-cell epitope; b) a trimer of wild-type 4-1BBL; and c) β2M;        and the heavy chain of Syn239/345 comprises: a) MHC heavy chain;        and b) IgG2a Fc. In Syn341/348 the first unit of the 4-1BBL        trimer comprises amino acids 50-254 of wild-type 4-1BBL; the        second and third units of the 4-1BBL trimer comprise amino acids        80 to 254 of wild-type 4-1BBL.    -   4) Syn341/349. The light chain of Syn341/349 comprises: an OVA        T-cell epitope; b) a trimer of amino acids 80-254 of 4-1BBL        comprising a K127A substitution in each unit of the trimer, with        a linker GlySerSerSerSer between the first and second units and        between the second and third units of the trimer; and c) β2M;        and the heavy chain of Syn239/345 comprises: a) MHC heavy chain;        and b) IgG2a Fc.

The resulting synTac heterodimers were cultured in vitro withovalbumin-specific T cells for 3 days or 5 days, at concentrations of 0,1, 3.17, 10.01, 31.65, and 100 nM synTac. Controls included: a) mediumalone; b) phorbol 12-myristate 13-acetate (PMA) and the ionophoreA23187; and c) an anti-CD3 antibody and an anti-CD28 antibody.

After 3 days, and after 5 days, the concentration of IFN-γ, IL-2, IL-6,TNF, IL-10, IL-17A, and IL-4 in the culture medium was determined. Inaddition, the viability of the OVA-specific T cells, and theproliferation of the OVA-specific T cells, was determined.

The data are depicted in FIGS. 38-46 .

As shown in FIG. 38 through FIG. 46 , Syn 341/349 induces production ofIL-2 (a cellular fitness cytokine); induces production of cytotoxiccytokines TNFα and IFN-γ; and also induces proliferation and enhancesviability of epitope-specific T cells.

Example 7: Production of synTacs in CHO Cells

SynTacs comprising wild-type (wt) 4-1BBL, or comprising 4-1BBL withamino acid substitutions as set out in FIG. 47 were transientlyexpressed in CHO cells. The amount of synTac produced was determined.The amounts produced are provided in FIG. 47 .

Example 8: In Vivo Effect of a 4-1BBL synTac

A synTac comprising a human papilloma virus (HPV) E7 antigenic peptideand a 4-1BBL K127A variant of the present disclosure (referred to as“CUE:4-1BBL (K127A)” in FIG. 48 ) was administered at 5 mg/kg byintraperitoneal (IP) injection into mice bearing flank engrafted HPV⁺TC-1 lung carcinoma. As a control, phosphate buffered saline (PBS) wasadministered to mice bearing the same tumor. As shown in FIG. 48 , tumorvolume was decreased in mice treated with CUE:4-1BBL (K127A), comparedto mice treated with PBS.

Example 9: In Vivo Effects of Co-Administration of a 4-1BBL synTac andan Immune Checkpoint Inhibitor

As depicted in FIG. 49 , co-administration of a 4-1BBL synTac of thepresent disclosure and an anti-PD1 antibody reduced tumor volume in amouse tumor model, and increased the percent of tumor infiltratinglymphocytes (TILs) that were granzyme B⁺.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-54. (canceled)
 55. A method of treating an individual having a cancerthat expresses a human papilloma virus-16 (HPV16) E7 antigen, the methodcomprising administering to the individual a combination of (i) aneffective amount of an immune checkpoint inhibitor that is an anti-PD-1antibody, and (ii) an effective amount of a protein that is a homodimerof two multimeric polypeptides, wherein each multimeric polypeptidecomprises: a) a first polypeptide comprising: i) a peptide comprising anHPV16 E7 epitope; and ii) a 02-microglobulin (β2M) polypeptide; and b) asecond polypeptide comprising: i) two copies of a variant IL-2polypeptide, each copy having at least 95% amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:8, where amino acid 42is an amino acid other than a phenylalanine, and where amino acid 16 isan amino acid other than a histidine, and wherein the variant IL2polypeptides exhibit reduced binding affinity to an IL-2 receptor (IL2R)comprising alpha, beta, and gamma polypeptides having amino acidsequences set forth in SEQ ID NOs:18-20, compared to the bindingaffinity of the IL-2 amino acid sequence set forth in SEQ ID NO: 1 forthe IL2R; ii) a major histocompatibility complex (MHC) class I heavychain polypeptide; and iii) an immunoglobulin (Ig) Fc polypeptide, andwherein the two multimeric polypeptides are joined to each other by oneor more disulfide bonds that join the Ig Fc polypeptide of onemultimeric polypeptide to the Ig Fc polypeptide of the other multimericpolypeptide, and wherein the immune checkpoint inhibitor and the proteinare administered at the same time or at different times.
 56. A method oftreating according to claim 55, wherein each multimeric polypeptidecomprises a peptide linker interposed between one or more of: a) theHPV16 E7 peptide and the β2M polypeptide; b) a first copy of the variantIL-2 polypeptide and a second copy of the variant IL-2 polypeptide; c)one of the two copies of the variant IL-2 polypeptide and the MHC classI heavy chain polypeptide; and d) the MHC class I heavy chainpolypeptide and the Ig Fc polypeptide.
 57. A method of treatingaccording to claim 56, wherein the HPV16 E7 peptide is from 7 to 16amino acids in length, wherein each variant IL-2 polypeptide has atleast 98% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:8, where amino acid 42 is an amino acid other than aphenylalanine, and where amino acid 16 is an amino acid other than ahistidine, wherein the β2M polypeptide has at least 95% sequenceidentity to amino acids 21 to 119 of SEQ ID NO:95, wherein the MHC classI heavy chain polypeptide has at least 95% sequence identity to theamino acid sequence set forth in SEQ ID NO:50; and wherein the Ig Fcpolypeptide comprises an amino acid sequence having at least about 95%amino acid sequence identity to the amino acid sequence set forth in SEQID NO:21.
 58. A method of treating according to claim 57, wherein aminoacid 42 of the variant IL-2 polypeptide is Ala and amino acid 16 of thevariant IL-2 polypeptide is Ala.
 59. A method of treating according toclaim 58, wherein each variant IL-2 polypeptide has the amino acidsequence set forth in SEQ ID NO:49.
 60. A method of treating accordingto claim 57, wherein the β2M polypeptide has at least 98% sequenceidentity to amino acids 21 to 119 of the amino acid sequence set forthin SEQ ID NO:95, and wherein the MHC class I heavy chain polypeptide hasat least 98% sequence identity to the amino acid sequence set forth inSEQ ID NO:50.
 61. A method of treating according to claim 59, whereinthe β2M polypeptide has at least 98% sequence identity to amino acids 21to 119 of the amino acid sequence set forth in SEQ ID NO:95, and whereinthe MHC class I heavy chain polypeptide has at least 98% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:50.
 62. Amethod of treating according to claim 60, wherein the Ig Fc polypeptidecomprises an amino acid sequence having at least about 98% amino acidsequence identity to SEQ ID NO:47, and wherein each multimericpolypeptide comprises a disulfide bond joining a Cys residue at aminoacid 12 of the β2M polypeptide and a Cys residue at amino acid 236 ofthe MHC class I heavy chain polypeptide.
 63. A method of treatingaccording to claim 61, wherein the Ig Fc polypeptide comprises an aminoacid sequence having at least about 98% amino acid sequence identity tothe amino acid sequence set forth in SEQ ID NO:47, and wherein eachmultimeric polypeptide comprises a disulfide bond joining a Cys residueat amino acid 12 of the β2M polypeptide and a Cys residue at amino acid236 of the MHC class I heavy chain polypeptide.
 64. A method of treatingaccording to claim 57, wherein: a) the first polypeptide of eachmultimeric polypeptide comprises, from N-terminus to C-terminus: i) theHPV16 E7 peptide; and ii) the β2M polypeptide; and b) the secondpolypeptide of each multimeric polypeptide comprises, from N-terminus toC-terminus: i) the first variant IL-2 polypeptide; ii) the secondvariant IL-2 polypeptide; iii) the MHC class I heavy chain polypeptide;and iv) the Ig Fc polypeptide, wherein each multimeric polypeptide caninclude one or more peptide linkers interposed between one or morecomponents of the first and second polypeptides.
 65. A method oftreating according to claim 57, wherein: a) the first polypeptide ofeach multimeric polypeptide comprises, from N-terminus to C-terminus: i)the HPV16 E7 peptide; and ii) the β2M polypeptide; and b) the secondpolypeptide of each multimeric polypeptide comprises, from N-terminus toC-terminus: i) the MHC class I heavy chain polypeptide; ii) the Ig Fcpolypeptide; iii) the first variant IL-2 polypeptide; and iv) the secondvariant IL-2 polypeptide; wherein each multimeric polypeptide caninclude one or more peptide linkers interposed between one or morecomponents of the first and second polypeptides.
 66. A method oftreating according to claim 60, wherein: a) the first polypeptide ofeach multimeric polypeptide comprises, from N-terminus to C-terminus: i)the HPV16 E7 peptide; and ii) the β2M polypeptide; and b) the secondpolypeptide of each multimeric polypeptide comprises, from N-terminus toC-terminus: i) the first variant IL-2 polypeptide; ii) the secondvariant IL-2 polypeptide; iii) the MHC class I heavy chain polypeptide;and iv) the Ig Fc polypeptide, wherein each multimeric polypeptide caninclude one or more peptide linkers interposed between one or morecomponents of the first and second polypeptides.
 67. A method oftreating according to claim 60, wherein: a) the first polypeptide ofeach multimeric polypeptide comprises, from N-terminus to C-terminus: i)the HPV16 E7 peptide; and ii) the β2M polypeptide; and b) the secondpolypeptide of each multimeric polypeptide comprises, from N-terminus toC-terminus: i) the MHC class I heavy chain polypeptide; ii) the Ig Fcpolypeptide; iii) the first variant IL-2 polypeptide; and iv) the secondvariant IL-2 polypeptide, wherein each multimeric polypeptide caninclude one or more peptide linkers interposed between one or morecomponents of the first and second polypeptides.
 68. A method oftreating according to claim 61, wherein: a) the first polypeptide ofeach multimeric polypeptide comprises, from N-terminus to C-terminus: i)the HPV16 E7 peptide; and ii) the β2M polypeptide; and b) the secondpolypeptide of each multimeric polypeptide comprises, from N-terminus toC-terminus: i) the first variant IL-2 polypeptide; ii) the secondvariant IL-2 polypeptide; iii) the MHC class I heavy chain polypeptide;and iv) the Ig Fc polypeptide, wherein each multimeric polypeptide caninclude one or more peptide linkers interposed between one or morecomponents of the first and second polypeptides.
 69. A method oftreating according to claim 61, wherein: a) the first polypeptide ofeach multimeric polypeptide comprises, from N-terminus to C-terminus: i)the HPV16 E7 peptide; and ii) the β2M polypeptide; and b) the secondpolypeptide of each multimeric polypeptide comprises, from N-terminus toC-terminus: i) the MHC class I heavy chain polypeptide; ii) the Ig Fcpolypeptide; iii) the first variant IL-2 polypeptide; and iv) the secondvariant IL-2 polypeptide, wherein each multimeric polypeptide caninclude one or more peptide linkers interposed between one or morecomponents of the first and second polypeptides.
 70. A method accordingto claim 55, wherein the protein is administered in an amount of from 1mg/kg to 5 mg/kg of body weight.
 71. A method according to claim 57,wherein the protein is administered in an amount of from 1 mg/kg to 5mg/kg of body weight.
 72. A method according to claim 58, wherein theprotein is administered in an amount of from 1 mg/kg to 5 mg/kg of bodyweight.
 73. A method according to claim 59, wherein the protein isadministered in an amount of from 1 mg/kg to 5 mg/kg of body weight. 74.A method according to claim 60, wherein the protein is administered inan amount of from 1 mg/kg to 5 mg/kg of body weight.
 75. A methodaccording to claim 61, wherein the protein is administered in an amountof from 1 mg/kg to 5 mg/kg of body weight.
 76. A method according toclaim 62, wherein the protein is administered in an amount of from 1mg/kg to 5 mg/kg of body weight.
 77. A method according to claim 63,wherein the protein is administered in an amount of from 1 mg/kg to 5mg/kg of body weight.
 78. A method according to claim 64, wherein theprotein is administered in an amount of from 1 mg/kg to 5 mg/kg of bodyweight.
 79. A method according to claim 65, wherein the protein isadministered in an amount of from 1 mg/kg to 5 mg/kg of body weight. 80.A method according to claim 66, wherein the protein is administered inan amount of from 1 mg/kg to 5 mg/kg of body weight.
 81. A methodaccording to claim 67, wherein the protein is administered in an amountof from 1 mg/kg to 5 mg/kg of body weight.
 82. A method according toclaim 68, wherein the protein is administered in an amount of from 1mg/kg to 5 mg/kg of body weight.
 83. A method according to claim 69,wherein the protein is administered in an amount of from 1 mg/kg to 5mg/kg of body weight.
 84. A method according to claim 57, wherein theimmune checkpoint inhibitor is nivolumab or pembrolizumab.